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https://openalex.org/W2735171399	https://europepmc.org/articles/pmc4934141?pdf=render	English	null	SfN Journals: Two Paths, One Goal: Sharing Strong Science	ENeuro	2,016	cc-by	860	SfN Journals: Two Paths, One Goal: Sharing Strong Science consultation processes is to provide a more rapid deci- sion to the authors, as well as a more focused idea of what would be needed for a revised manuscript to be appropriate for publication. Most of our readers know that The Society for Neuroscience now publishes two journals, The Journal of Neuroscience and eNeuro, but many are puzzled about the difference between them and which journal would be more appropriate for a specific submission. While the journals will be evolving con- tinually, and more differences are likely to appear over time, we would like to start to outline the similarities and differ- ences between the two journals here. Structural and Functional The first difference between the two Society for Neuro- science journals is structural: eNeuro is a fully open- access, on-line journal. eNeuro publishes articles upon acceptance and does not have formal volumes or issues. JNeurosci maintains a print edition that is published weekly, and the on-line issue parallels the print version. Articles in JNeurosci are available freely 6 months after publication, and authors can pay an additional fee to make their article open access immediately. The second difference is functional: eNeuro can be more experimental in the review process and try new methods to increase the rapidity and transparency of review. Editorial Board and Content At both journals, all editors are respected, working neu- roscientists who publish articles in areas related to those they handle, and who, therefore, have firsthand knowl- edge publishing articles in neuroscience journals today. In terms of content, JNeurosci focuses on mechanistic stud- ies that provide in-depth understanding of novel findings in neuroscience. JNeurosci has no upper or lower limit for the number of figures, but the reviewers and editors are asked to identify the most significant findings of the man- uscript and to evaluate how the findings will change think- ing in the neuroscience field. eNeuro has no constraints on the length of the paper or the number of figures, and is interested in publishing novel, but not necessarily mech- anistic, findings that are of potential broad interest. As part of the process of differentiating the role of the two journals, the Brief Communication format, which focuses on important new observations that do not yet have a mechanistic underpinning, has been moved from JNeu- rosci to eNeuro. Along with important novel observations in neuroscience, eNeuro encourages the submission of papers on new methods, commentaries, negative results, important replication, and failure to replicate studies. Im- portantly, eNeuro does not accept papers that would require 3 months of work to be appropriate for publica- tion, so if many more experiments are required, the paper might be recommended for re-review as a more in depth study at either journal. Peer Review The system for peer review is different at eNeuro and JNeurosci, but both journals have a procedure for coming to consensus, particularly when reviewers have disparate views about a manuscript. The review process at eNeuro is innovative and involves a double-blind procedure that maintains the anonymity of both the reviewers and the authors. Identifying information is removed before the review process, and authors are asked to eliminate infor- mation from the body of the manuscript that identifies the laboratory. After the manuscript is reviewed, reviewers are invited into a consensus review process mediated by the Reviewing Editor to make sure that the decision is reached as a full consensus between reviewers and edi- tor. The Reviewing Editor then provides a review summary laying out exactly what changes or experiments would be needed for acceptance, or why the manuscript was re- jected. The Journal of Neuroscience uses a tiered review process in which a Reviewing Editor invites the reviewers, evaluates the reviews, and makes a recommendation on whether or not the manuscript is acceptable for publica- tion to one of the Senior Editors. The Senior Editors handle papers from overlapping subsets of the Reviewing Editors, providing a level of consistency for decisions on manuscripts across broader areas of neuroscience. In- creasingly, the editors at JNeurosci are using a consulta- tion process adapted from that used by eNeuro to reach consensus between reviewers with differing recommen- dations, or to consult among editors about manuscripts that are not sent out for formal review. The goal of these Editorial July/August 2016, 3(4) e0154-16.2016 1–1 Summary Although JNeurosci and eNeuro publish studies across the breadth of the neuroscience field and are focused on technical excellence, the format, review process, and types of articles published already differ substantially between the two journals. We anticipate that JNeurosci and eNeuro will continue to evolve, both by strategic decisions based on ongoing discussion between the editorial boards of the jour- nals, and as a result of the natural evolution that occurs as authors choose where to submit their work. Both journals welcome input from the neuroscience community on how to provide options for publishing rigorous and insightful re- search, and on how these two Society for Neuroscience journals can best serve the field. Christophe Bernard, EiC, eNeuro Marina Picciotto, EiC, JNeurosci DOI:10.1523/ENEURO.0154-16.2016 Christophe Bernard, EiC, eNeuro Marina Picciotto, EiC, JNeurosci DOI:10.1523/ENEURO.0154-16.2016 July/August 2016, 3(4) e0154-16.2016 1–1
https://openalex.org/W4389147063	https://edumediasolution.com/society/article/download/221/101	Indonesian	null	Pengelolaan Jagung Menjadi Susu Bubuk Jagung Agar Meningkatkan Nilai Jual Jagung Pada Desa Bolo Kecamatan Madapangga Kabupaten Bima	Society	2,023	cc-by-sa	2,574	Society: Jurnal Pengabdian Masyarakat, Vol. 2, No. 1 (2023): Januari, pp. 44-48 https://edumediasolution.com/index.php/society E-ISSN:2827-878X (Online -Elektronik) Society: Jurnal Pengabdian Masyarakat, Vol. 2, No. 1 (2023): Januari, pp. 44-48 https://edumediasolution.com/index.php/society E-ISSN:2827-878X (Online -Elektronik) ARTICLE INFO Sweet Corn Milk is milk produced by processing sweet corn where because the sugar content of it has been used in it then the use of sugar will certainly be much less even if the bias is not used at all. Sweet Corn Milk is very beneficial for our metabolism for both children and adults and seniors. Compared to ordinary milk the content of vitamins and minerals is also not much different and has also had its own color of corn used which is a bit yellowish so it gives rise to an appetite to drink it. Corn milk is obtained by grinding corn seeds that have been boiled in water. The mill results are filtered to obtain filtrate which is then pasteurized and flavored to enhance the taste. The content of carbohydrate extracts in corn milk is influenced by corn varieties, the amount of water added, the length of time and storage conditions, the smoothness of the mill, and the heat treatment. The advantages of corn milk compared to cow's milk or soybeans are the raw materials are easy to come by at a not very high price. Corn does not contain lactate intolerance (which makes milk smell fishy). Corn milk contains more fiber, suitable for those who diet. Some corn benefits our health. A Rich Source of Caloric, Prevention of Bowel Cancer and Hemorrhoids, A Vitamin-Rich Source, Contains The Necessary Minerals, Antioxidant Properties of Corn, Protects the Heart, Prevents Anemia, Lowers Bad Cholesterol, Protection against Diabetes and Hypertension. If we process with a little food processing technique into corn milk then it will be worth more with different appearances and flavors that can be customized to the taste of consumers. So it is expected that with this activity mothers and people who participate in the activity can obtain knowledge and information on how to make corn milk. Corn milk can be consumed alone or sold to supplement the family’s income. Article History Received : Dec 2, 2022 Revised : Dec 20, 2022 Accepted : Jan 11, 2023 Article History Received : Dec 2, 2022 Revised : Dec 20, 2022 Accepted : Jan 11, 2023 Pengelolaan Jagung Menjadi Susu Bubuk Jagung Agar Meningkatkan Nilai Jual Jagung Pada Desa Bolo Kecamatan Madapangga Kabupaten Bima M. Rimawan a,1,, Ismunandar a,2, Aliah Pratiwi a,3 Intisariharyanti a,4 Ita Iftitah a,5 Alwi a,6 a Sekolah Tinggi Ilmu Ekonomi Bima, Jl. Woltermonginsidi No 1, Kota Bima 84113, Indonesia 1 rimawan111@gmail.com ; 2 andar.stiebima@gmail.com ; 3 aliahpratiwi@ymail.com 4 intisariharyanti@gmail.com 5 itaiftitah.stiebima@gmail.com , alwi_muhammad1317@yahoo.co.id * corresponding author: rimawan111@gmail.com A. Penduhuluan Kondisi ekonomi yang diharapkan Indonesia adalah ekonomi yang berkelanjutan dan memiliki beberapa sektor sebagai pilar maupun penopang kegiatan ekonominya. Pertumbuhan yang tinggi tercermin dari kompetisi individu-individu dalam menciptakan inovasi. Ekonomi kreatif yang didalamnya terdapat industri-industri kreatif memiliki daya tawar yang tinggi di dalam ekonomi berkelanjutan karena individu-individunya memiliki modal kreativitas yang mereka gunakan untuk menciptakan inovasi yang bisa memberikan nilai tambah ekonomi (Afifah et al., 2022; Sunreni et al., 2022). Ekonomi kreatif menjadi salah satu konsep untuk pengembangan perekonomian di Indonesia yang mana, Indonesia bisa mengembangkan model, ide dan talenta dari rakyat untuk dapat 44 Society: Jurnal Pengabdian Masyarakat, Vol. 2, No. 1 (2023): Januari, pp. 44-48 https://edumediasolution.com/index.php/society E-ISSN:2827-878X (Online -Elektronik) menginovasi dan menciptakan suatu hal. Pola pikir kreatif sangat diperlukan untuk tetap tumbuh berkembang serta bertahan di masa yang akan dating (Lase, 2022). menginovasi dan menciptakan suatu hal. Pola pikir kreatif sangat diperlukan untuk tetap tumbuh berkembang serta bertahan di masa yang akan dating (Lase, 2022). Jagung merupakan salah satu bahan makanan pokok yang memiliki kedudukan penting setelah beras bagi masyarakat Indonesia. Selain itu, jagung juga merupakan sumber bahan baku bagi sektor industri termasuk industri pangan. Kandungan gizi penting pada jagung adalah karbohidrat dan lemak. Karbohidrat jagung terdiri dari pati, gula, serat kasar dan pentosan. Pati jagung terdiri dari amilosa dan amilopektin, sedangkan gulanya berupa sukrosa. Disamping kaya phytochemical juga memberikan perlindungan terhadap berbagai penyakit kronis. Berikut ini beberapa manfaat jagung bagi kesehatan kita. Sumber yang Kaya Kalorin, Pencegahan Kanker Usus dan Wasir, Sumber yang Kaya Vitamin, Mengandung Mineral yang Diperlukan, Sifat antioksidan Jagung, Melindungi Jantung, Mencegah Anemia, Menurunkan Kolesterol Jahat, Perlindungan terhadap Diabetes dan Hipertensi. (Syamsir, 2008). Sebagian besar produksi jagung di daerah ini hanya dijual dalam bentuk segar yang biasanya dipergunakan untuk direbus ataupun dijadikan sebagai sayuran dan sebagiannya lagi dikeringkan untuk dijadikan sebagai pakan ternak. Tanaman jagung di daerah ini belum diupayakan dan dikembangkan dengan baik, belum ada diversifikasi pengembangan tanaman jagung menjadi produk olahan yang berdaya jual tinggi dan bernilai ekonimis. Jagung sangat bermanfaat jika ditinjau dari nilai gizinya dan jagung mudah didapat baik dikota maupun didesa. Namun jika hanya mengolah jagung dengan direbus, digoreng atau dikukus maka konsumen kurang tertarik, dan menganggapnya sudah biasa. Jika kita olah dengan sedikit teknik pengolahan pangan menjadi susu jagung maka akan bernilai lebih dengan penampilan yang berbeda dan rasa yang dapat disesuaikan dengan selera konsumen. A. Penduhuluan Sehingga diharapkan dengan adanya kegiatan ini ibu-ibu serta masyarakat yang ada di Desa Bolo bisa melanjutkan kegiatan ini dan dapat memperoleh pengetahuan dan informasi bagaimana membuat susu jagung tersebut. Susu Jagung ini dapat dikonsumsi sendiri atau dijual untuk menambah income keluarga. Perkembangan zaman semakin pesat dan dalam era industri 4.0 ini kita selaku pemuda-pemudi di tuntut untuk berpikir kreatif dan bisa menguasai teknologi, dan mampu melihat peluang yang ada. Pemuda harus bisa menjadikan ketrampilan yang mereka miliki untuk menyongsong kemajuan daerahnya masing-masing dengan melihat potensi yang ada di wilayah tersebut. Jagung merupakan potensi yang ada di desa Bolo kecamatan Madapangga yang hanya disajikan dengan direbus, dibakar dan digoreng, Potensi jangung yang ada di Desa Bolo bisa dioptimalkan lagi apabila dijadikan sebagai bahan olahan Susu agar memiliki nilai tambah, namun dalam hal ini perlu adanya kerja sama antara pemerintah dengan pemuda dan mahasiswa untuk memberikan literasi terkait bagaimana cara membuat olahan makanan dari jagung menjadi Susu Jagung atau seperti snack kekinian yang dapat meningkatkan kemandirian masyarakat dari sisi perekonomian. B. Kajian Literatur Melimpahnya ketersediaan jagung di berbagai daerah di Indonesia membuka peluang kegiatan usaha bagi masyarakat. Salah satunya adalah dengan membuat produk olahan dari jagung. Pengolahan jagung dapat meningkatkan nilai tambah dan nilai jualnya. Pengembangan produk olahan jagung masih sangat potensial karena makin besar peluang pasar yang dapat dicapai. Hal tersebut dapat membuka peluang bagi pengembangan usaha industri aneka produk olahanjagung di Indonesia (Hambali, Suryani, & Ihsanur, 2006). Demikian juga halnya dengan potensi pengembangan produk olahan jagung manis. Mengingat potensinya yang sangat besar ini maka diperlukan usaha bersama untuk mengembangkan usaha ini sehingga ke depan dapat bersaing dengan usaha yang lebih besar. Jagung manis juga memiliki keunggulan tertentu, jagung dapat berkhasiat sebagai pengobatan, sehingga dapat berperan juga sebagai pangan fungsional, karena dapat berperan dalam mencegah dan 45 Society: Jurnal Pengabdian Masyarakat, Vol. 2, No. 1 (2023): Januari, pp. 44-48 https://edumediasolution.com/index.php/society E-ISSN:2827-878X (Online -Elektronik) mengurangi resiko penyakit seperti diabetes melitus, jantung, kolesterol, dan lain-lain. Sehingga dengan makin meningkatnya tuntutan terhadap kualitas hidup, maka tuntutan terhadap konsumsi makanan yang berperan dalam pengobatan juga semakin tinggi, oleh karena itulah maka perlu dilakukan pelatihan teknologi pengolahan produk jagung manis kepada masyarakat sehingga pada saat over produksi dapat dilakukan diversifikasi pengolahan menjadi aneka produk olahan jagung manis. Hasil pertanian yang ingin mereka olah menjadi produk olahan adalah jagung manis, karena di sekitar tempat tinggal mereka di Kecamatan Denpasar Timur, banyak petani yang menanam jagung manis. Jagung manis tersebut hanya dijual langsung ke pengepul atau dibawa langsung ke pasar, sehingga harga jualnya sangat murah, hanya Rp. 2.500 – Rp. 4.500 per kg. Jika diolah menjadi produk olahan, seperti menjadi susu jagung bisa mendapat keuntungan bersih Rp 30.000 –Rp.35.000 per kg jagung manis. Pengolahan produk akan dapat meningkatkan nilai jual dan nilai tambahnya. C. Metode Kegiatan pengabdiaan pada masyarakat ini termaksud dalam salah satu program kerja kegiatan Kuliah Kerja nyata (KKN) yang berlokasi di desa bolo kecamatan madapangga Kabupaten Bima pada tahun 2022. Sebelum melakukan Program Pengabdian Masyarakat ini, pelaksana kegiatan melakukan pendampingan secara intensif. Melakukan persiapan tempat, alat dan bahan sekaligus materi tentang wirausaha dan pembuatan susu jagung. Pengabdian ini dalam proses kegiatannya melalui beberapa tahap: a. Survey Awal Kegiatan survey dilakukan pada potensi jagung di Desa Bolo, dimana sebagian Masyarakat disana petani jagung. Dimana pada saat panen langsung menjual ke pengepul, dijual dengan cara di bakar, direbus. b. Sosialisasi Program Sosialisasi program dilaksanakan setelah tahap survey telah dilakukan yaitu dengan melakukan pertemuan dengan petani jagung, masyarakat desa Bolo yang mengikuti pelatihan pemanfaatan jagung untuk dijadikan susu bubuk jangung yang bisa dijadikan sebagai pemacu untuk meningkatkan pendapatan rumah tangga, serta meningkatkan perekonomian desa dan menjadi desa mandiri c. Pelatihan Peserta pelatihan dikumpulkan di Kantor Desa Bolo, para peseerta diajarkan bagaimana cara membuat susu jagung, d. Evaluasi Evaluasi dilakukan setelah semua tahap diatas telah terlaksana yaitu dengan meminta kritik dan saran melalui Tanya jawab pada peserta pelatihan mengenai pelaksanaan program. Serta produk akhirnya di bazarkan di Desa Bolo D. Results and Discussion Sosialisasi Tahap Ketiga yaitu pelatihan diberikan kepada masyrakat di desa bolo, yang akan menjadi mitra nanti dan akan melanjutkan kegitan ini setelah kami selesai kkn adapaun dalam pelatihan ini masyrakat diberikan sedikit terkait pemahaman nilai jual jagung ini dan seetelah itu diberikan catatan bagai mana bahan-bahan dan cara pembutannya sampai proses pengepakan / pengemasan serta penjualan secara online. Pelatihan ini yang menjadi pemateri adalah anak-anak mahsiswa kkn. Tahap Ketiga yaitu pelatihan diberikan kepada masyrakat di desa bolo, yang akan menjadi mitra nanti dan akan melanjutkan kegitan ini setelah kami selesai kkn adapaun dalam pelatihan ini masyrakat diberikan sedikit terkait pemahaman nilai jual jagung ini dan seetelah itu diberikan catatan bagai mana bahan-bahan dan cara pembutannya sampai proses pengepakan / pengemasan serta penjualan secara online. Pelatihan ini yang menjadi pemateri adalah anak-anak mahsiswa kkn. Gambar 2 : Pelatihan Pembutan susu jagung Tahap Empat Evaluasi dilakukan setelah pelatihan denagn cara melihat hasil kerja Masyarakat desa Bolo dan Mitra yang melanjutkan setelah kegiatan. Alhamdulilah setelah melakukna pelatihan masysrakat sudah bisa melakuan kegiatan produksi sendiri bahkan ada sudah memasarkan di pasar tradisionla dan lewat penjualan online, masih menggunkan Whatapps dan IG Gambar 2 : Pelatihan P Gambar 2 : Pelatihan Pembutan susu jagung Gambar 2 : Pelatihan Pembutan susu jagung Gambar 2 : Pelatihan Pembutan susu jagung Tahap Empat Evaluasi dilakukan setelah pelatihan denagn cara melihat hasil kerja Masyarakat desa Bolo dan Mitra yang melanjutkan setelah kegiatan. Alhamdulilah setelah melakukna pelatihan masysrakat sudah bisa melakuan kegiatan produksi sendiri bahkan ada sudah memasarkan di pasar tradisionla dan lewat penjualan online, masih menggunkan Whatapps dan IG Tahap Empat Evaluasi dilakukan setelah pelatihan denagn cara melihat hasil kerja Masyarakat desa Bolo dan Mitra yang melanjutkan setelah kegiatan. Alhamdulilah setelah melakukna pelatihan masysrakat sudah bisa melakuan kegiatan produksi sendiri bahkan ada sudah memasarkan di pasar tradisionla dan lewat penjualan online, masih menggunkan Whatapps dan IG Tahap Empat Evaluasi dilakukan setelah pelatihan denagn cara melihat hasil kerja Masyarakat desa Bolo dan Mitra yang melanjutkan setelah kegiatan. Alhamdulilah setelah melakukna pelatihan masysrakat sudah bisa melakuan kegiatan produksi sendiri bahkan ada sudah memasarkan di pasar tradisionla dan lewat penjualan online, masih menggunkan Whatapps dan IG D. Results and Discussion Selama beberapa hari melaksanakan program kerja, kami memperoleh hasil dari pengamatan di lapangan bahwa hampir semua pelaku UMKM pembuatan Kerupuk belum melakukan inovasi pada produk mereka dan dalam hal menjual produk belum memiliki Kemasan sehingga belum bisa tembus ke pasar Modern. Hal ini terjadi karena kurangnya pengetahuan masyarakat desa Tonggorisa tentang cara berbisnis yang tepat. Selain itu para pelaku UKM juga kesulitan dalam melakukan penjualan produk Kerupuk kecuali yang hanya dijual dipasar tradisional dan dipasarkan keliling desa. Tahap Pertama Kegiatan survey dilakukan pada potensi jagung di Desa Bolo, dimana sebagian Masyarakat disana petani jagung. Dimana pada saat panen langsung menjual ke pengepul, dijual dengan cara di bakar, direbus. Antusiasme warga sangat membantu kesuksesan kegiatan pada tahap pertama ini. Tahap Kedua Sosialisasi program dilaksanakan setelah tahap survey telah dilakukan yaitu dengan melakukan pertemuan dengan petani jagung, masyarakat desa Bolo yang mengikuti pelatihan pemanfaatan jagung untuk dijadikan susu bubuk jangung yang bisa dijadikan sebagai pemacu untuk 46 Society: Jurnal Pengabdian Masyarakat, Vol. 2, No. 1 (2023): Januari, pp. 44-48 https://edumediasolution.com/index.php/society E ISSN 2827 878X (O li El k ik) meningkatkan pendapatan rumah tangga, serta meningkatkan perekonomian desa dan menjadi desa mandiri meningkatkan pendapatan rumah tangga, serta meningkatkan perekonomian desa dan menjadi desa di i meningkatkan pendapatan rumah tangga, serta meningkatkan perekonomian desa dan menjadi desa mandiri Gambar 1. Sosialisasi Tahap Ketiga yaitu pelatihan diberikan kepada masyrakat di desa bolo, yang akan menjadi mitra nanti dan akan melanjutkan kegitan ini setelah kami selesai kkn adapaun dalam pelatihan ini masyrakat diberikan sedikit terkait pemahaman nilai jual jagung ini dan seetelah itu diberikan catatan bagai mana bahan-bahan dan cara pembutannya sampai proses pengepakan / pengemasan serta penjualan secara online. Pelatihan ini yang menjadi pemateri adalah anak-anak mahsiswa kkn. meningkatkan pendapatan rumah tangga, serta meningkatkan perekonomian desa dan menjadi desa mandiri Gambar 1. Sosialisasi Tahap Ketiga yaitu pelatihan diberikan kepada masyrakat di desa bolo, yang akan menjadi mitra nanti dan akan melanjutkan kegitan ini setelah kami selesai kkn adapaun dalam pelatihan ini masyrakat diberikan sedikit terkait pemahaman nilai jual jagung ini dan seetelah itu diberikan catatan bagai mana bahan-bahan dan cara pembutannya sampai proses pengepakan / pengemasan serta penjualan secara online. Pelatihan ini yang menjadi pemateri adalah anak-anak mahsiswa kkn. Gambar 1. Sosialisasi Gambar 1. E. Simpulan Adanya kemauan dari pihak Desa Bolo dalam mendukung kegitan ini sehingga masyarakat Bolo sudah bisa memahami inovasi terkiat hasil alam Jagung menjadi Susu Jagung. Kelebihan pelaksanaan 47 Society: Jurnal Pengabdian Masyarakat, Vol. 2, No. 1 (2023): Januari, pp. 44-48 https://edumediasolution.com/index.php/society E-ISSN:2827-878X (Online -Elektronik) pengabdian ini yaitu materi sosialisasi dan pelatihan yang dapat menambah wawasan, pengetahuan, dan keterampilan guna menunjang pemasaran produk UMKM. Adanya inovasi produk diharapkan bisa meningkatkan penjulan dan pendapatan. Kekurangan pelaksanaan pengabdian yaitu adanya beberapa pelaku UMKM yang kurang menguasai perkembangan teknologi saat ini seperti penggunaan media sosial. Daftar Pustaka Anonim, 2006. Bahan Tambahan Pangan (Food Additive). Ebookpangan.Com. Anonim, 2006. Bahan Tambahan Pangan (Food Additive). Ebookpangan.Com. A. E. Setiyono, N. Ngatimun, and T. Musriati, “Pemanfaatkan Potensi Lokal Melalui Pembuatan Susu Jagung Guna Mencegah Stunting Pada Desa Gejugan,” J. Abdi Panca Mara, vol. 1, no. 1, pp. 18–21, 2020, doi: 10.51747/abdipancamarga.v1i1.639 p g B. Satiarini, “KAJIAN PRODUKSI DAN PROFITABILITAS PEMBUATAN SUSU JAGUNG,” pp. 1–23, 2006 Gardjito, M.Hadiwiyoto, S. 1994. Teori dan Prosedur Pengujian Mutu Susu dan Hasil Olahannya. Liberty. Yogyakarta. Hasbullah, 2001, Kripik Jagung, TeknologiTepat Guna Agroindustri, DIP Teknologi dan Industri, Sumatra Barat T. Larosta, “Susu jagung manis edamame,” 2020 Lase, A. (2022). Pelatihan dan Praktek Pembuatan Sabun Cuci Sunlight di Desa Onozalukhu, Kecamatan Lahewa, Kabupaten Nias Utara. Zadama: Jurnal Pengabdian Masyarakat, 1(1), 1– 6. https://doi.org/10.56248/zadama.v1i1. 12 p g ini et al, “Khasiat ‘Si Biji Manis Bertongkol’ Menjadi Susu Sebagai Alternatif Penambah Asam Folat Selama Kehamilan,” Semin. Nas. XI Pendidik. Biol. FKIP UNS, pp. 241–244, 2005 Pradiani, T. (2018). Pengaruh Sistem Pemasaran Digital Marketing Terhadap Peningkatan Volume Penjualan Hasil Industri Rumahan. Jurnal Ilmiah Bisnis Dan Ekonomi Asia, 11(2), 46–53. https://doi.org/10.32812/jibeka.v11i2.45 p g j Soekarto, S. 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https://openalex.org/W2080339831	https://europepmc.org/articles/pmc3589260?pdf=render	English	null	A Novel Lipopeptide from Skin Commensal Activates TLR2/CD36-p38 MAPK Signaling to Increase Antibacterial Defense against Bacterial Infection	PloS one	2,013	cc-by	6,993	Abstract Staphylococcus epidermidis (S.epidermidis) plays important protective roles by directly producing or by stimulating hosts to produce antimicrobial peptides (AMPs) against pathogenic infections. Although several AMPs from S.epidermidis have been identified, molecules that stimulate hosts to produce AMPs remain largly unknown. Here we demonstrate that a new lipopeptide (named LP01) purified from S.epidermidis culture media has a unique structure with heneicosanoic acid (21 carbons) binding to lysine11 of a peptide chain. In vitro LP01 increased the expression of b-defensin 2(hBD2) and hBD3 in neonatal human epidermal keratinocytes(NHEK), leading to increased capacity of cell lysates to inhibit the growth of S.aureus. In vivo LP01 induced the expression of mouse b-defensin 4(mBD4) to decrease the survival of local S.aureus in skin and systemic S.aureus survival in liver. The induction of beta-defensins by LP01 was dependent on TLR2 as Tlr2-deficient mice had decreased mBD4. Furthermore, knockdown of CD36 decreased the expression of hBD2 and hBD3, and p38 MAPK inhibitor significantly inhibited the expression of hBDs induced by LP01.Taken together, these findings demonstrate that lipopeptide LP01 from normal commensal S.epidermidis increases antimicrobial peptide hBD2 and hBD3 expression via the activation of TLR2/CD36-p38 MAPK, thus enhancing antimicrobial defense against pathogenic infections. Citation: Li D, Lei H, Li Z, Li H, Wang Y, et al. (2013) A Novel Lipopeptide from Skin Commensal Activates TLR2/CD36-p38 MAPK Signaling to Increase Antibacterial Defense against Bacterial Infection. PLoS ONE 8(3): e58288. doi:10.1371/journal.pone.0058288 Editor: Bernhard Ryffel, French National Centre for Scientific Research, France Editor: Bernhard Ryffel, French National Centre for Scientific Research, France Received November 25, 2012; Accepted February 1, 2013; Published March 5, 2013 Copyright: 2013 Li 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: 2013 Li et al. This is an open-access article distributed under the terms of the Creative Commons Attrib use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by National Natural Science Foundation of China grants (NSFC) 81072422, 31222021&31170867, grants 11QA1401900, 12ZZ039 to Yuping Lai, NSFC grant 31100109 to Deming Jiang, Shanghai Natural science Foundation grant11ZR1409900 to Meiling Zhang, the program for professor of Special Appointment (Eastern Scholar) at Shanghai Institutions (Yuping Lai), and the Science and Technology Commission of Shanghai Municipality grant 11DZ2260300. A Novel Lipopeptide from Skin Commensal Activates TLR2/CD36-p38 MAPK Signaling to Increase Antibacterial Defense against Bacterial Infection Dongqing Li, Hu Lei, Zhiheng Li, Hongquan Li, Yue Wang, Yuping Lai* Shanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, People’s Republic of China ghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China Normal University, Shanghai, People’s Republic of Chin Abstract Part of in-vitro studies was also supported by Johnson and Johnson. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Part of in-vitro studies was also supported by Johnson and Johnson. East China Normal University has applied for the pending Chinese patent for LP01 (Name: The preparation and application of lipopeptide 01 and its derivatives; Application Number: 201210138021.0; and date of patent: May 7, 2012)). There are no further patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: yplai@bio.ecnu.edu.cn Besides S.epidermidis, several other bacteria have been shown to produce lipopeptides to help hosts against pathogenic infections by disturbing gram-positive bacterial cell wall synthesis [6]. One of the most well-established lipopeptides with direct antimicrobial activity is daptomycin [6]. This lipopeptide can destruct the membrane of Gram-positive pathogens including Methicillin- resistant Staphylococcus aureus. In addition to daptomycin, other lipopeptides including Iturin A [7] and Fengycin [8] from Bacillus subtilis, polymyxins from Paenibacillus polymyxa [9] and fusaricidins from Paenibacillus polymyxa [10] have been identified to exert their direct antimicrobial activity. Besides direct antimicrobial activity, synthetic lipopeptides such as MALP-2 can induce antimicrobial peptide expression in several cell types such as keratinocytes [5]. These observations thereby raise the possibility that the molecule from S.epidermidis to induce AMP might be a lipopeptide. Bacterial Strains and Mice Staphylococcus epidermidis 1457, Staphylococcus aureus, Escherichia coli DH5a and Propionibacterium acnes were stored in our laboratory. S.epidermidis 1457 and S.aureus were cultured at 37uC for 16 h in Tryptic Soy Broth (TSB) medium (Sigma, St Louis, MO). E.coli DH5a was cultured at 37uC for 16 h in Lysogeny Broth (LB) medium. P.acnes was culture at 37uC in Reinforced Clostridial Medium in anaerobic pouch (MGC, JAPAN). All mice were kept under specific pathogen free conditions and maintained in accordance with the institutional guidelines. All animal experi- ments were approved by East China Normal University Animal Care and Use Committee. Figure 1. The identification of the lipopeptide from Staphylococcus epidermidis. A. The analysis of the lipopeptide from S.epidermidis by thin-layer chromatography. Duplicate samples were loaded on TLC plate and molecules with different hydrophobicity were separated. Water was used to show hydrophobicity of lipopeptides and ninhydrin was used to show that the lipopeptide contains amino acids. The arrows indicate LP01. B. Q-TOF MS/MS analysis of lipopeptide in positive-ion model. Q-TOF MS/MS analysis showed that the amino acid sequence of the lipopeptide was DIISTIGDLVKWIIDTVIIDATE. C&D. Two possible structures of the lipopeptide. Aspartic acid (D1) at N-terminus and lysine (K11) are two amino acids with free NH3 +, and free -COOH of heneicosanoic acid might react with NH3 + to form CO–NH. Thereby heneicosanoic acid may bind to 1D or 11K of the peptide chain. d i 10 1371/j l 0058288 001 doi:10.1371/journal.pone.0058288.g001 methanol at 37uC for overnight and dried by rotary evaporation followed by loading on HPLC with a C18 column for further purification. The purified lipopeptide was analyzed by thin-layer chromatography (TLC) on silica-coated glass plates in solvent system (butyl alcohol: acetic acid: H2O = 4:2:1, v/v/v). Protein sequences were analyzed by Q-TOF MS/MS de-novo sequencing (Waters, ACQUITYTM UPLC&Q-TOF Premier). The structure of lipid of the lipopeptide was analyzed by GC/MS (Shimadzu, GC/MS-QP2010). lipopeptide induced AMP against S.aureus infection. Our findings reveal the potential use of commensal bacterium-derived lipopep- tides in treatment of skin infections. Introduction Skin, as a first line of defense, interfaces with the environment and is exposed to a myriad of microbes. Among these microbes, Staphylococcus epidermidis (S.epidermidis) is the most abundant bacte- rium that resides on skin and generally has a benign relationship with its host. Previously, we have gained considerable insights into beneficial roles of S.epidermidis by balancing inflammatory re- sponses after skin injury. We discovered that lipoteichoic acid from S.epidermidis substantially attenuated keratinocyte response to skin injury through a TLR2-dependent inhibition of the TLR3 signaling via TNF receptor-associated factor 1 (TRAF1), thus suppressing unwanted inflammatory cytokine production [1]. Besides the regulation of inflammation in skin injury, other groups showed that S.epidermidis produced some antimicrobial molecules including Staphylococcin 1580 [2], Pep5 [3], PSMs [4] to benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens. We have also demonstrated that less than 10 kDa molecules from S.epidermidis culture media induced the production of b-defensins to enable the skin to mount an enhanced response to pathogens [5]. However, the identity of the molecule from S.epidermidis to induce b-defensins remains unknown. Given that skin commensal bacteria increase host defense against pathogenic infections and lipopeptides from bacteria exert direct antimicrobial activity or induce host to produce antimicro- bial peptides, we hypothesized that the molecule from S.epidermidis to induce b-defensins might be a kind of lipopeptide. In this study we successfully purified one novel lipopeptide from S.epidermidis culture media and further delineated the mechanism by which the March 2013 \| Volume 8 \| Issue 3 \| e58288 1 March 2013 \| Volume 8 \| Issue 3 \| e58288 PLOS ONE \| www.plosone.org Lipopeptide Increases Antibacterial Host Defense Figure 1. The identification of the lipopeptide from Staphylococcus epidermidis. A. The analysis of the lipopeptide from S.epidermidis by thin-layer chromatography. Duplicate samples were loaded on TLC plate and molecules with different hydrophobicity were separated. Water was used to show hydrophobicity of lipopeptides and ninhydrin was used to show that the lipopeptide contains amino acids. The arrows indicate LP01. B. Q-TOF MS/MS analysis of lipopeptide in positive-ion model. Q-TOF MS/MS analysis showed that the amino acid sequence of the lipopeptide was DIISTIGDLVKWIIDTVIIDATE. C&D. Two possible structures of the lipopeptide. Aspartic acid (D1) at N-terminus and lysine (K11) are two amino acids with free NH3 +, and free -COOH of heneicosanoic acid might react with NH3 + to form CO–NH. Real-time Quantitative RT-PCR Total RNA was prepared using Trizol Reagent (TaKaRa, Japan) following the manufacturer’s instructions. RNA was quantified by Thermo NANODROP 2000 spectrophotometer. Total RNA (1 mg) was reverse transcribed using PrimeScriptH RT reagent Kit (TaKaRa, Japan) according to the manufacturer’s instructions. Real-time RT-PCR was conducted on Mx3005P (Stratagene, USA) using SYBRH Premix ExTaq (TaKaRa, Japan). The quantification of gene expression was determined by the comparative 2DDCT method. The primers used in this manu- scriptare shown in the following: mBD4 forward: GGCTTCAGT- CAT GAGGATCCAT; mBD4 reverse: TTTGGGTAAAGGCTGCAAGTG; mBD14 forward: GTGGC CGGTGTGCTGTACT; mBD14 reverse: CGCTATTAGA ACATCGACCTATTTGT; hBD2 forward: CCAGCCAT- CAGCCATGAGGGT; hBD2 reverse: GGAGCCCTTTCT- Total RNA was prepared using Trizol Reagent (TaKaRa, Japan) following the manufacturer’s instructions. RNA was quantified by Thermo NANODROP 2000 spectrophotometer. Total RNA (1 mg) was reverse transcribed using PrimeScriptH RT reagent Kit (TaKaRa, Japan) according to the manufacturer’s instructions. Real-time RT-PCR was conducted on Mx3005P (Stratagene, USA) using SYBRH Premix ExTaq (TaKaRa, Japan). The quantification of gene expression was determined by the comparative 2DDCT method. The primers used in this manu- scriptare shown in the following: mBD4 forward: GGCTTCAGT- CAT GAGGATCCAT; mBD4 reverse: Introduction Thereby heneicosanoic acid may bind to 1D or 11K of the peptide chain. doi:10.1371/journal.pone.0058288.g001 Primary Cell Culture and Stimulation by Lipopeptide Neonatal human epidermal keratinocytes (Cascade Biologics, USA) were cultured in EpiLife medium supplemented with EDGS, 0.06 mM CaCl2 (Cascade Biologics, USA) and Pen Strep(100 units/ml Penicillin and 100 mg/ml Sreptomycin). Murine primary keratinocytes were isolated from newborn skin by using dispase II (Sigma, St Louis, MO) and cultured in 154CF medium supplemented with HKGS and 0.2 mM CaCl2 and Pen Strep(100 units/ml Penicillin and 100 mg/ml Sreptomycin) (Invitrogen, Shanghai). Keratinocytes were seeded in 6-, 12- or Lipopeptide Purification and Identification All the assays were per- formed in triplicate and repeated at least two times. GAATCCGCA; hBD3 forward: GCCTCTT CCAGGTGTTTTTG; hBD3 reverse: GAGACCACAGGTGC- CAATTT. The relative expression levels were determined by normalizing expression to 18s rRNA or glyceraldehyde 3- phosphate dehydrogenase (GAPDH). All the assays were per- formed in triplicate and repeated at least two times. 24-well plates to grow to 70% confluence. To test whether the induction of hBDs by lipopeptide was in a dose-dependent manner, 2.5, 5, 10, 15, 20, 25, 30 mg/ml lipopeptide was used to stimulate NHEK cells. 15 mg/ml lipopeptide was used to stimulate murine primary keratinocytes. 24 h later, cells were harvested. The expression of genes was analyzed by using real-time RT-PCR and the protein levels of hBDs were determined by hBD2 and hBD3 ELISA Kit (Peprotech, Hamburg, Germany). Lipopeptide Purification and Identification S.epidermidis 1457 was grown in TSB medium at 37uC for overnight. Next day, the overnight culture was diluted 1:100 into TSB and grown for another 16 hours. Bacterial supernatants were collected and filtered by 0.22 mm stericup. Bacterium-free culture supernatant was adjusted to pH = 2.0 and then stored at 4uC for overnight. Next day, the crude lipopeptide was extracted by March 2013 \| Volume 8 \| Issue 3 \| e58288 2 PLOS ONE \| www.plosone.org Lipopeptide Increases Antibacterial Host Defense Figure 2. LP01 induces b-defensin expression in primary keratinocytes. A&B. Quantification of hBD2 and hBD3 mRNA expression in NHEKs treated with LP01 or SECM. C&D. hBD2 and hBD3 protein expression in cell lysates of NHEKs treated with LP01 by ELISA. E&F. Quantification of mBD4 and mBD14 mRNA expression stimulated with 15 mg/mL LP01 in primary murine keratinocytes. Primary murine keratinocytes were isolated from C57BL/6 mice. G&H. Quantification of hBD2 and hBD3 mRNA expression in NHEKs stimulated by LP01 with shortened peptide chain. I&J. Quantification of hBD2 and hBD3 mRNA expression in NHEKs stimulated by LP01 with shortened fatty acid chain. P,0.05, P,0.01 and *P,0.001, n.s., no significance. P values were determined by one-way ANOVA or two-tailed t test. Data are the means 6 SEM of n = 3 and representative of two independent experiments. doi:10.1371/journal.pone.0058288.g002 Figure 2. LP01 induces b-defensin expression in primary keratinocytes. A&B. Quantification of hBD2 and hBD3 mRNA expression in NHEKs treated with LP01 or SECM. C&D. hBD2 and hBD3 protein expression in cell lysates of NHEKs treated with LP01 by ELISA. E&F. Quantification of mBD4 and mBD14 mRNA expression stimulated with 15 mg/mL LP01 in primary murine keratinocytes. Primary murine keratinocytes were isolated from C57BL/6 mice. G&H. Quantification of hBD2 and hBD3 mRNA expression in NHEKs stimulated by LP01 with shortened peptide chain. I&J. Quantification of hBD2 and hBD3 mRNA expression in NHEKs stimulated by LP01 with shortened fatty acid chain. P,0.05, P,0.01 and P,0.001, n.s., no significance. P values were determined by one-way ANOVA or two-tailed t test. Data are the means 6 SEM of n = 3 and representative of two independent experiments. doi:10 1371/journal pone 0058288 g002 p p doi:10.1371/journal.pone.0058288.g002 p p doi:10.1371/journal.pone.0058288.g002 GAATCCGCA; hBD3 forward: GCCTCTT CCAGGTGTTTTTG; hBD3 reverse: GAGACCACAGGTGC- CAATTT. The relative expression levels were determined by normalizing expression to 18s rRNA or glyceraldehyde 3- phosphate dehydrogenase (GAPDH). Immunoblotting Normal human epidermal keratinocytes were treated with 15 mg/mL of LP01 for 1 hour in the presence or absence of inhibitors. Cells were lysed in the RIPA buffer (pH 7.4) containing protease inhibitor cocktail (Roche, Pleasanton, CA) after washed by ice-cold PBS for 3 times. Protein concentrations of the extracts were measured by BCATM Protein Assay Kit (Novagen, San Diego, CA). 30 mg of total protein was used for western blot. The lysates were separated by 12% SDS-PAGE and analyzed by immunoblotting with phosphorylated p38 MAPK antibody or p38 MAPK antibody (Cell signaling, Danvers, MA), respectively. The membrane was scanned by Odyssey machine (Li-Cor Biosciences, Lincoln, NE). S.aureus Infection in vivo The backs of 8-week adult mice were shaved and hair was removed by chemical depilation. 2 mg/kg of LP01 and PBS was intradermally injected into mouse back, respectively. Next day, 50 mL of live S.aureus (OD600 = 0.7–0.8) complexed with cytodex beads (Sigma, St Louis, MO) as carrier was intradermally injected 2 hours after injection of LP01. Lesional sizes caused by S.aureus infection were measured daily. At day 3, skin around the lesional sites, liver and spleen were taken and homogenized for S.aureus culture. Inactivation of TLR2 or p38 MAPK TLR2 inhibitor OxPAPC (InvivoGen, San Diego, CA) or p38 MAPK inhibitor SB202190(Sigma, St Louis, MO) was added 10 minutes before normal human keratinocytes or murine primary keratinocytes were treated with 15 mg/ml of LP01. 24 hours later, total RNA was extracted and cDNA synthesized by the PrimeScriptH RT reagent Kit (TaKaRa, Japan). The level of gene expression was quantified by real-time RT-PCR by using Stratagene Mx3005P. Lipopeptide Increases Antibacterial Host Defense Lipopeptide Increases Antibacterial Host Defense concentration of the lysates was determined by BCATM Protein Assay Kit (Novagen, San Diego, CA). 10 mg of cell lysates was incubated with 16106 CFU P.acnes, S.aureus or S.epidermidis at 37uC for 3 h. The bacterial were then serially diluted by 10-fold with PBS and plated on RCM agar plates (P.acnes), TSB agar plates (S.aureus, S.epidermidis) or LB agar plates (E.coli DH5a). Statistical Analysis All data are present as mean6SEM. Two-tailed t-test was used to determine significances between two groups. The significances among multiple groups were determined by One-way or Two-way ANOVA with Bonferroni post test of GraphPad Prism Version 5 (San Diego, CA). For all statistical tests, we considered P values ,0.05 to be statistically significant. Bacterial Killing Assay To test the capacity of NHEK cell lysate stimulated with LP01 in the inhibition of the growth of bacteria, NHEK cells were cultured in no P/S Epilife medium and then were treated with 15 mg/mL of LP01. After 24 hours, 100 mL of phosphate buffered saline (PBS) containing protease inhibitor cocktail was added into each well. Cells were collected by cell scraper and then sonicated on ice-cold water. After removed cell debris by centrifugation, the March 2013 \| Volume 8 \| Issue 3 \| e58288 PLOS ONE \| www.plosone.org 3 The Structure of Lipopeptide 01 (LP01) from Skin Commensal Staphylococcus epidermidis We have shown that less than 10 kDa molecules from S.epidermidis culture media induced the production of b-defensins and that one synthetic lipopeptide such as MALP-2 induced hBDs expression in keratinocytes [5], we thus hypothesized that the Figure 3. LP01 increases antibacterial activity of keratinocytes. The growth of S.aureus (A), E.coli DH5a (B), P.acnes (C) and S.epidermidis (D) after exposure to lysates of LP01-treated neonatal human epidermal keratinocytes or the growth of S.aureus after exposure to cell culture medium of LP01-treated NHEKs (E). The bacterial were serially diluted 10-fold with PBS and then counted by colony formation. P,0.05; n.s., no significance. P values were determined by t-test. All data are representative of three independent experiments with n = 3 and are means 6 SEM. doi:10.1371/journal.pone.0058288.g003 Figure 3. LP01 increases antibacterial activity of keratinocytes. The growth of S.aureus (A), E.coli DH5a (B), P.acnes (C) and S.epidermidis (D) after exposure to lysates of LP01-treated neonatal human epidermal keratinocytes or the growth of S.aureus after exposure to cell culture medium of LP01-treated NHEKs (E). The bacterial were serially diluted 10-fold with PBS and then counted by colony formation. P,0.05; n.s., no significance. P values were determined by t-test. All data are representative of three independent experiments with n = 3 and are means 6 SEM. doi:10.1371/journal.pone.0058288.g003 March 2013 \| Volume 8 \| Issue 3 \| e58288 PLOS ONE \| www.plosone.org 4 Lipopeptide Increases Antibacterial Host Defense Figure 4. LP01 protects mice from S.aureus infection. A. Photograph of skin lesions caused by S.aureus at 3 days after S.aureus injection. B. ImageJ analysis of the lesional size of A. C. ImageJ analysis of the lesional size of PBS- or scrambled lipopeptide-treated mice. Local S.aureus survival in skin (D and E) and systemic S.aureus survival in liver (F) and spleen (G) of PBS- or scrambled lipopeptide- or LP01- pretreated mice. P,0.01; P,0.001. P values were determined by two-tailed t test or two-way ANOVA. All data are the means 6 SEM of n = 6 and representative of two independent experiments. doi:10.1371/journal.pone.0058288.g004 Figure 4. LP01 protects mice from S.aureus infection. A. Photograph of skin lesions caused by S.aureus at 3 days after S.aureus injection. B. ImageJ analysis of the lesional size of A. C. ImageJ analysis of the lesional size of PBS- or scrambled lipopeptide-treated mice. The Structure of Lipopeptide 01 (LP01) from Skin Commensal Staphylococcus epidermidis Local S.aureus survival in skin (D and E) and systemic S.aureus survival in liver (F) and spleen (G) of PBS- or scrambled lipopeptide- or LP01- pretreated mice. P,0.01; **P,0.001. P values were determined by two-tailed t test or two-way ANOVA. All data are the means 6 SEM of n = 6 and representative of two independent experiments. doi:10.1371/journal.pone.0058288.g004 4 (mBD4, mouse homolog of human hBD2) but not mBD14 (mouse homolog of human hBD3) in primary murine keratino- cytes (Fig. 2E and 2F). Furthermore, the capacity of LP01 to induce hBDs was more dependent on the intact peptide chain than the intact fatty acid. This was made evident by the fact that a shortened peptide chain with heneicosanoic acid markedly decreased hBDs induction while the intact peptide chain with shortened fatty acid slightly decreased hBDs induction (Fig. 2 G– J). These data suggest that LP01 with heneicosanoic acid binding to peptide chain (22 amino acids) is the lipopeptide from S.epidermidis to induce beta-defensins. molecule from Staphylococcus epidermidis to induce b-defensins might be a lipopetide. To test this, we used methanol extraction, acid precipitation, thin-layer chromatography and high-performance liquid chromatography (HPLC) to isolate lipopeptides from the S.epidermidis culture media(Fig. 1) and confirmed that the lipopeptide we purified from S.epidermidis culture media had the capacity to induce huaman beta-defensin 2 (hBD2) and hBD3 expression in neonatal human epidermal keratinocytes (NHEKs)(- data not shown). The de-novo peptide sequencing showed that the peptide chain of the lipopeptide is molecule from Staphylococcus epidermidis to induce b-defensins might be a lipopetide. To test this, we used methanol extraction, acid precipitation, thin-layer chromatography and high-performance liquid chromatography (HPLC) to isolate lipopeptides from the S.epidermidis culture media(Fig. 1) and confirmed that the lipopeptide we purified from S.epidermidis culture media had the capacity to induce huaman beta-defensin 2 (hBD2) and hBD3 expression in neonatal human epidermal keratinocytes (NHEKs)(- data not shown). The de-novo peptide sequencing showed that the peptide chain of the lipopeptide is DIISTIGDLVKWIIDTVIIDATE(Fig. 1B), and Gas chromatog- raphy mass spectrometry revealed that this lipopeptide contained one heneicosanoic acid C20H40COOH(Fig. 1C and 1D). The analysis of the peptide sequence showed that aspartic acid (D1) at N-terminus and the amino acid at site 11 lysine (K11) might be amino acids where heneicosanoic acid bound to the peptide chain. Therefore, structures of the lipopeptide might be as shown in Fig. 1C and Fig. 1D. LP01 Increases Antibacterial Activity against S.aureus Infection It is known that human hBD2 and hBD3 from epidermal keratinocytes exert bactericidal activity against E.coli and S.aureus infection [11,12]. Since LP01 significantly induced hBD2 and hBD3 protein expression in kerationcytes (Fig. 2A–2D) but not too much in cell culture media (data not shown), we next examined whether the induction of hBD2 and hBD3 by LP01 could increase the antibacterial capacity of keratinocytes in vitro. A cell lysate of undifferentiated NHEKs pretreated with 10 mg/ml of LP01 significantly inhibited the growth of S.aureus (Fig. 3A), but did not inhibit E.coli (Fig. 3B), P.acnes (Fig. 3C), and S.epidermidis itself (Fig. 3D). Moreover, due to the low secreted hBD2 and hBD3, cell To determine the structure-function relationship of the lipope- tides, we synthesized the above two possible lipopeptides LP01 and LP78 and then evaluated their capacity to induce the expression of antimicrobial peptides. Compared to S.epidermidis culture media (SECM), synthetic LP01 significantly increased both mRNA and protein of hBD2 and hBD3 in NHEKs (Fig. 2A–2D) while synthetic LP78 did not (data not shown). In addition to NHEKs, LP01 significantly increased the expression of mouse beta-defensin March 2013 \| Volume 8 \| Issue 3 \| e58288 PLOS ONE \| www.plosone.org 5 Figure 5. The induction of beta-defensins by LP01 is dependent on TLR2. A, B. The expression of hBD2 and hBD3 in NHEKs treated with 15 mg/mL of LP01 in the presence or absence of TLR2 inhibitor OxPAPC. C. mBD4 expression in mouse ears 24 h after injection by 2 mg/kg of LP01. D. Photograph of skin lesions caused by S.aureus at 3 days after S.aureus injection in Tlr2+/+ and Tlr2–/– mice. E. S.aureus survival in skin of PBS- and LP01- pretreated Tlr2+/+ and Tlr2–/– mice. F–G. Quantification of hBD2 and hBD3 expression in NHEK cells stimulated with LP01 after CD14 or CD36 was silenced. P,0.05, **P,0.001. P values were determined by one-way or two-way ANOVA. All data are representative of two independent experiments with n = 3–6 and are the means 6 SEM. doi:10.1371/journal.pone.0058288.g005 Lipopeptide Increases Antibacterial Host Defense Lipopeptide Increases Antibacterial Host Defense Figure 5. The induction of beta-defensins by LP01 is dependent on TLR2. A, B. The expression of hBD2 and hBD3 in NHEKs treated with 15 mg/mL of LP01 in the presence or absence of TLR2 inhibitor OxPAPC. C. mBD4 expression in mouse ears 24 h after injection by 2 mg/kg of LP01. D. LP01 Increases Antibacterial Activity against S.aureus Infection Photograph of skin lesions caused by S.aureus at 3 days after S.aureus injection in Tlr2+/+ and Tlr2–/– mice. E. S.aureus survival in skin of PBS- and LP01- pretreated Tlr2+/+ and Tlr2–/– mice. F–G. Quantification of hBD2 and hBD3 expression in NHEK cells stimulated with LP01 after CD14 or CD36 was silenced. P,0.05, *P,0.001. P values were determined by one-way or two-way ANOVA. All data are representative of two independent experiments with n = 3–6 and are the means 6 SEM. doi:10.1371/journal.pone.0058288.g005 (Fig. 4F and 4G). Taken together, these results demonstrate that the LP01 from S. epidermidis increases host defense aganist S. aureus infection. culture media of NHEKs treated with LP01 was not able to inhibit the growth of S.aureus (Fig. 3E). To confirm that the induction of antimicrobial peptides in keratinocytes by LP01 would be relevant to protection against S.aureus infection in vivo, LP01 was intradermally injected into mice 24 and 2 hours before an infectious challenge at the site with S.aureus. LP01-treated mice showed significantly smaller infectious skin lesions when compared with control mice injected with PBS or scrambled lipopeptide (Fig. 4A–4C). Accompanied with smaller infectious lesions, the survival of S.aureus at the local site of infection was significantly decreased (Fig. 4D and 4E). In addition, the survival of S.aureus in liver, but not in spleen, was decreased The Activation of p38 MAPK is Required for the Induction of Beta-defensins by LP01 The Activation of p38 MAPK is Required for the Induction of Beta-defensins by LP01 we first used the TLR2 inhibitor OxPAPC to block TLR2 activation. OxPAPC completely inhibited the expression of hBD2 and hBD3 induced by LP01 (Fig. 5A and 5B). Consistent with our in vitro observations, LP01 lost its capacity to induce the expression of mBD4 in Tlr2-deficient mice compared to wild-type mice (Fig. 5C). In addition, LP01 failed to protect mice from S.aureus infection in Tlr2-deficient mice as shown with bigger infectious skin lesions and increased surviving S.aureus (Fig. 5D and 5E). Furthermore, knockdown of a coreceptor for TLR2 heterodimer, CD36, significanlty decreased hBD2 and hBD3 expression induced by LP01 (Fig. 5F and 5G). However, knockdown of antoher coreceptor for TLR2 heterodimer, CD14, only significantly decreased LP01-induced hBD2 expres- sion, but not hBD3 expression (Fig. 5F and 5G). Altogether, these data demonstrate that lipopeptide LP01 activates TLR2 and CD36 to induce hBD2 and hBD3 in skin. we first used the TLR2 inhibitor OxPAPC to block TLR2 activation. OxPAPC completely inhibited the expression of hBD2 and hBD3 induced by LP01 (Fig. 5A and 5B). Consistent with our in vitro observations, LP01 lost its capacity to induce the expression of mBD4 in Tlr2-deficient mice compared to wild-type mice (Fig. 5C). In addition, LP01 failed to protect mice from S.aureus infection in Tlr2-deficient mice as shown with bigger infectious skin lesions and increased surviving S.aureus (Fig. 5D and 5E). Furthermore, knockdown of a coreceptor for TLR2 heterodimer, CD36, significanlty decreased hBD2 and hBD3 expression induced by LP01 (Fig. 5F and 5G). However, knockdown of antoher coreceptor for TLR2 heterodimer, CD14, only significantly decreased LP01-induced hBD2 expres- sion, but not hBD3 expression (Fig. 5F and 5G). Altogether, these data demonstrate that lipopeptide LP01 activates TLR2 and CD36 to induce hBD2 and hBD3 in skin. It has been reported that activation of TLR2 regulates multiple downstream molecules including NF-kB and mitogen-activated protein kinases(MAPKs) [15]. Specifically, p38 MAPK has been shown to play an important role in hBD2 and hBD3 production in epithelial cells such as keratinocytes [16,17]. We thus hypothesized that p38 MAPK might be the downstream molecule of TLR2 involved in the induction of hBDs by LP01. To test this hypothesis, we first checked if LP01 induced p38 MAPK phosphorylation after TLR2 was blocked. TLR2 inhibitor OxPAPC markedly decreased the phosphorylation of p38MAPK (Thr180/Tyr182) by LP01 (Fig. 6A and 6B). The Induction of Beta-defensins by LP01 is Dependent on TLR2 and CD36 Having established the role of LP01 in S.aureus skin infection, we next sought to explore the mechanism by which LP01 regulates antimicrobial peptide expression. Since TLR2 is a well- known receptor on keratinocytes for lipopeptides [13,14], we assumed that the LP01 isolated from S.epidrmidis would activate TLR2 to induce antimicrobial peptide expression. To test this, March 2013 \| Volume 8 \| Issue 3 \| e58288 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Lipopeptide Increases Antibacterial Host Defense Figure 6. The activation of p38 MAPK is required for the induction of beta-defensins by LP01. A&B. TLR2 inhibitor OxPAPC inhibited p38 phosphorylation induced by 15 mg/mL of LP01 in NHEK cells. C&D. p38 MAPK inhibitor SB202190 inhibited p38 phosphorylation induced by 15 mg/ mL of LP01 in NHEK cells. E&F. p38 MAPK inhibitor SB202190 completely inhibited hBD2 and hBD3 expression induced by 15 mg/mL of LP01 in NHEK cells. G. p38 MAPK inhibitor SB202190 completely inhibited mBD4 expression induced by 15 mg/mL of LP01 in murine primary keratinocytes. P,0.001. P values were determined by one-way ANOVA. Data are the means 6 SEM of n = 3 and representative of two to three independent experiments. doi:10.1371/journal.pone.0058288.g006 Figure 6. The activation of p38 MAPK is required for the induction of beta-defensins by LP01. A&B. TLR2 inhibitor OxPAPC inhibited p38 phosphorylation induced by 15 mg/mL of LP01 in NHEK cells. C&D. p38 MAPK inhibitor SB202190 inhibited p38 phosphorylation induced by 15 mg/ mL of LP01 in NHEK cells. E&F. p38 MAPK inhibitor SB202190 completely inhibited hBD2 and hBD3 expression induced by 15 mg/mL of LP01 in NHEK cells. G. p38 MAPK inhibitor SB202190 completely inhibited mBD4 expression induced by 15 mg/mL of LP01 in murine primary keratinocytes. **P,0.001. P values were determined by one-way ANOVA. Data are the means 6 SEM of n = 3 and representative of two to three independent experiments. doi:10.1371/journal.pone.0058288.g006 p doi:10.1371/journal.pone.0058288.g006 Discussion q p y Toll-like receptors (TLRs) have been reported to play an important role in host defense. They can recognize pathogen- associated molecular patterns (PAMPs) from microbes and damge- associated molecular patterns (DAMPs) from hosts. TLR2, as one of TLR family members, ususally works as TLR2/TLR1 or TLR2/TLR6 heterodimer and recognize a broad range of ligands, especially ligands of cell-wall componenets from gram- positive bacteria such as lipoteichoic acid (LTA) as well as peptidoglycan and lipoproteins [27]. LP01, as a lipopeptide from S.epidermidis, binds to TLR2 and then activates p38 MAPK to induce hBD2 and hBD3 expression. Furthrmore, we have shown that the coreceptor CD36 is also involved in the induction of hBD2 and hBD3 by LP01, which is consistent with previous observation that CD36 facilitates TLR2 recognition [28]. How- ever, although it is reported that both CD36 and CD14 are required for TLR2 in response to LTA [29,30,31], in our system the silencing of CD14 only slightly decreased hBD2 expression but not hBD3 while knockdown of CD36 significantly reduced the expression of both hBD2 and hBD3. One explanation of this phenomenon is that CD36 and CD14 participate in different TLR2 signaling complex as CD36 is specifically involved in TLR2/TLR6-mediated response but not TLR2/TLR1-mediated response [29] while CD14 usually enhances signaling in respose to TLR2/TLR1 ligand Pam3CysSK4 [32]. The other explanation is that CD14 usually facilitates TLR2 ligands binding to TLR2 and NF-kB activation [32]. However, our data show that the induction of hBD2 and hBD3 requires p38 MAPK activation but not NF-kB activation. This is probably the reason that silencing of CD14 does not decrease the induction of hBD2 and hBD3 by LP01. Altogether, these data suggest that LP01, as a lipopeptide, activates TLR2/TLR6 signaling other than TLR2/TLR1 signaling. However, further investigation is needed. To date, most studies on skin microbes have been conducted to analyze the type and diversity of microbes present on the skin by classic culture techniques or identified by sequencing surveys of 16s rRNA. However, sparse attention has been paid to the beneficial function of these microbes that inhabit on our skin. Here we have identified one new lipopeptide (LP01) from S.epidermidis, one of the most commonly isolated bacterial species from healthy human skin [18,19], and show that the lipopeptide activates TLR2/CD36-p38 MAPK to induce hBD2 and hBD3 expression, thus enabling the skin to mount an enhanced antimicrobial defense against pathogenic infections. Discussion Our results reveal that skin commensals play an important role in host antimicrobial defense and suggest that the preservation of these commensals on skin may be an effective way to achieve maintenance of healthy ‘normal’ skin function. Epidemiologic and clinical studies indicate that the increased incidence of autoimmune and allergic diseases in developed countries is associated with reduced microbial exposure and alteration of microbial communities in various body sites [20]. Although high-throughput metabolomics analyses have been used to identify core microbial communities linked to the onset of pathologies, there is still a big challenging to define a ‘normal healthy’ microbiota of skin at the functional level [21,22,23]. Moreover, the molecular mechanisms underlying microbe-host interactions that shape host immune functions remain largely unknown. Previous work from Schittek and our group has demonstrated that skin commensals induce antimicrobial peptide RNase7 to amplify the innate immune response [24] and that S.epidermidis conditioned culture medium (SECM) increases anti- microbial peptide expression in keratinocytes [5]. Here we further advance our understanding of how skin commensal S.epidermidis regulates host immune responses against bacterial infections by defining the structure of lipopeptide LP01 from S.epidermidis and delineating the mechanism by which LP01 induces the expression of antimicrobial peptide hBD2 and hBD3. In conclusion, these findings suggest that skin resident commensals are necessary for our protection from infections. Specifically, we find that lipopeptide LP01 from S.epidermidis exerts its effect by enhancing antimicrobial defense via the activation of TLR2/CD36-p38 MAPK signaling. Furthermore, our results indicate that identification of specific molecules from skin commensals to understand its roles in shaping host immune response is not only of primary importance for human health, but will also lead to the development of more rational antibacterial approaches. Our results also emphasize disadvantages of in- discriminate use of topical and systemic antibiotics to treat skin infections. In order to identify the structure of LP01, we used Q-TOF mass spectrometry de-novo sequencing technique to identify the amino acid sequence of the peptide chain and gas chromatography mass spectrometry technique to identify the fatty acid chain as priviously described [25,26]. According to the amino acid sequence of the lipopeptide, we found that there were two possiblities for fatty acid heneicosanoic acid binding to peptide chain. One possibility was that heneicosanoic acid bound to Lys11, the other was that heneicosanoic acid bound to N-terminal amino acid Asp1. 1. Lai Y, Di Nardo A, Nakatsuji T, Leichtle A, Yang Y, et al. (2009) Commensal bacteria regulate Toll-like receptor 3-dependent inflammation after skin injury. Nat Med 15: 1377–1382. Acknowledgments We thank Xi Li from Worldwide EM Innovation Center, Jonhson &Johnson China Ltd. for helpful discussion. Discussion To determine structure-function relationship, the above two possible lipopeptides were synthesized and their capacity to induce hBD2 and hBD3 expression was tested. Our results demonostrate that only the lipopeptide with heneicosanoic acid binding to Lys11 has the capacity to induce hBD2 and hBD3 expression, suggesting that Lys11 is the binding site of heneicosa- noic acid. Furthermore, we have synthesized several derivatives of LP01 to prove that the intact peptide chain is essensial for the function of LP01 to induce AMPs. Our data show that heneicosanoic acid binding to shortened peptide chain completely 2. Fitzgerald RJ, Morhart RE, Marquez C, Adams BO (1986) Inhibition of caries in hamsters treated with staphylococcin 1580. Infect Immun 54: 288–290. Lipopeptide Increases Antibacterial Host Defense Lipopeptide Increases Antibacterial Host Defense that p38 MAPK is the critical downstream molecule of TLR2 to induce hBD2 and hBD3 expression induced by LP01. that p38 MAPK is the critical downstream molecule of TLR2 to induce hBD2 and hBD3 expression induced by LP01. lost its capacity to induce hBD2&3 while shortened fatty acid chain binding to the intact peptide chain kept its capacity to induce AMPs. These data suggest that the lipopeptide only with a unique structure has the capacity to induce AMPs. Author Contributions Conceived and designed the experiments: YL DL. Performed the experiments: DL H. Lei ZL H. Li YW. Analyzed the data: DL H. Lei YL. Wrote the paper: YL DL. Conceived and designed the experiments: YL DL. Performed the experiments: DL H. Lei ZL H. Li YW. Analyzed the data: DL H. Lei YL. Wrote the paper: YL DL. The Activation of p38 MAPK is Required for the Induction of Beta-defensins by LP01 In addition to OxPAPC, p38 MAPK inhibitor, SB202190 completely inhibited p38 MAPK phosphor- ylation (Fig. 6C and 6D). Furthermore, the inhibition of p38 MAPK significantly decreased the expression of hBD2 and hBD3 in NHEKs (Fig. 6E and 6F) as well as mBD4 in primary murine keratinocytes (Fig. 6G). Taken together, these results demonstrate PLOS ONE \| www.plosone.org March 2013 \| Volume 8 \| Issue 3 \| e58288 7 Lipopeptide Increases Antibacterial Host Defense 3. Sahl HG, Ersfeld-Dressen H, Bierbaum G, Josten M, Kordel M, et al. (1987) Different mechanisms of insensitivity to the staphylococcin-like peptide Pep 5. Zentralbl Bakteriol Mikrobiol Hyg A 267: 173–185. 17. Lan CC, Wu CS, Huang SM, Kuo HY, Wu IH, et al. High-Glucose Environment Inhibits p38MAPK Signaling and Reduces Human beta-Defensin- 3 Expression [corrected] in Keratinocytes. Mol Med 17: 771–779. 18. Evans CA, Smith WM, Johnston EA, Giblett ER (1950) Bacterial flora of the normal human skin. J Invest Dermatol 15: 305–324. 4. Cogen AL, Yamasaki K, Sanchez KM, Dorschner RA, Lai Y, et al. Selective antimicrobial action is provided by phenol-soluble modulins derived from Staphylococcus epidermidis, a normal resident of the skin. J Invest Dermatol 130: 192–200. normal human skin. J Invest Dermatol 15: 305–324 19. Marples MJ (1969) The normal flora of the human skin. Br J Dermatol 81: Suppl 1: 2–13. 5. Lai Y, Cogen AL, Radek KA, Park HJ, Macleod DT, et al. (2010) Activation of TLR2 by a small molecule produced by Staphylococcus epidermidis increases antimicrobial defense against bacterial skin infections. J Invest Dermatol 130: 2211–2221. 20. Okada H, Kuhn C, Feillet H, Bach JF (2010) The ‘hygiene hypothesis’ for autoimmune and allergic diseases: an update. Clin Exp Immunol 160: 1–9. 21. Costello EK, Lauber CL, Hamady M, Fierer N, Gordon JI, et al. (2009) Bacterial community variation in human body habitats across space and time. Science 326: 1694–1697. 6. Tally FP, DeBruin MF (2000) Development of daptomycin for gram-positive infections. J Antimicrob Chemother 46: 523–526. 22. Qin J, Li R, Raes J, Arumugam M, Burgdorf KS, et al. (2010) A human gut microbial gene catalogue established by metagenomic sequencing. Nature 464: 59–65. 7. Klich MA, Arthur KS, Lax AR, Bland JM (1994) Iturin A: a potential new fungicide for stored grains. Mycopathologia 127: 123–127. 23. Hormannsperger G, Clavel T, Haller D (2012) Gut matters: microbe-host interactions in allergic diseases. J Allergy Clin Immunol 129: 1452–1459. 8. Vanittanakom N, Loeffler W, Koch U, Jung G (1986) Fengycin–a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J Antibiot (Tokyo) 39: 888–901. 24. Wanke I, Steffen H, Christ C, Krismer B, Gotz F, et al. Skin commensals amplify the innate immune response to pathogens by activation of distinct signaling pathways. J Invest Dermatol 131: 382–390. ( y ) 9. Lipopeptide Increases Antibacterial Host Defense Dixon RA, Chopra I (1986) Polymyxin B and polymyxin B nonapeptide alter cytoplasmic membrane permeability in Escherichia coli. J Antimicrob Che- mother 18: 557–563. 25. Guo Y, Huang E, Yuan C, Zhang L, Yousef AE Isolation of a Paenibacillus sp. strain and structural elucidation of its broad-spectrum lipopeptide antibiotic. Appl Environ Microbiol 78: 3156–3165. 10. Li J, Jensen SE (2008) Nonribosomal biosynthesis of fusaricidins by Paenibacillus polymyxa PKB1 involves direct activation of a D-amino acid. Chem Biol 15: 118–127. pp 26. Kim PI, Bai H, Bai D, Chae H, Chung S, et al. (2004) Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. J Appl Microbiol 97: 942–949. 11. Harder J, Bartels J, Christophers E, Schroder JM (2001) Isolation and characterization of human beta -defensin-3, a novel human inducible peptide antibiotic. J Biol Chem 276: 5707–5713. J pp 27. Baranova IN, Kurlander R, Bocharov AV, Vishnyakova TG, Chen Z, et al. (2008) Role of human CD36 in bacterial recognition, phagocytosis, and pathogen-induced JNK-mediated signaling. J Immunol 181: 7147–7156. 12. Harder J, Bartels J, Christophers E, Schroder JM (1997) A peptide antibiotic from human skin. Nature 387: 861. 28. Bjorkbacka H (2006) Multiple roles of Toll-like receptor signaling in atherosclerosis. Curr Opin Lipidol 17: 527–533. 13. Schroder NW, Morath S, Alexander C, Hamann L, Hartung T, et al. (2003) Lipoteichoic acid (LTA) of Streptococcus pneumoniae and Staphylococcus aureus activates immune cells via Toll-like receptor (TLR)-2, lipopolysaccharide- binding protein (LBP), and CD14, whereas TLR-4 and MD-2 are not involved. J Biol Chem 278: 15587–15594. 29. Hoebe K, Georgel P, Rutschmann S, Du X, Mudd S, et al. (2005) CD36 is a sensor of diacylglycerides. Nature 433: 523–527. 30. Stuart LM, Deng J, Silver JM, Takahashi K, Tseng AA, et al. (2005) Response to Staphylococcus aureus requires CD36-mediated phagocytosis triggered by the COOH-terminal cytoplasmic domain. J Cell Biol 170: 477–485. 14. Sumikawa Y, Asada H, Hoshino K, Azukizawa H, Katayama I, et al. (2006) Induction of beta-defensin 3 in keratinocytes stimulated by bacterial lipopeptides through toll-like receptor 2. Microbes Infect 8: 1513–1521. COOH-terminal cytoplasmic domain. J Cell Biol 170: 477–485. 31. Reed-Geaghan EG, Savage JC, Hise AG, Landreth GE (2009) CD14 and toll- like receptors 2 and 4 are required for fibrillar A{beta}-stimulated microglial activation. J Neurosci 29: 11982–11992. 15. Vasselon T, Hanlon WA, Wright SD, Detmers PA (2002) Toll-like receptor 2 (TLR2) mediates activation of stress-activated MAP kinase p38. References 2. Fitzgerald RJ, Morhart RE, Marquez C, Adams BO (1986) Inhibition of caries in hamsters treated with staphylococcin 1580. Infect Immun 54: 288–290. March 2013 \| Volume 8 \| Issue 3 \| e58288 PLOS ONE \| www.plosone.org 8 Lipopeptide Increases Antibacterial Host Defense J Leukoc Biol 71: 503–510. J 32. Nilsen NJ, Deininger S, Nonstad U, Skjeldal F, Husebye H, et al. (2008) Cellular trafficking of lipoteichoic acid and Toll-like receptor 2 in relation to signaling: role of CD14 and CD36. J Leukoc Biol 84: 280–291. 16. Kanda N, Watanabe S (2008) Leptin enhances human beta-defensin-2 production in human keratinocytes. Endocrinology 149: 5189–5198. 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https://openalex.org/W3006393912	https://www.researchsquare.com/article/rs-7358/latest.pdf	English	null	Resting Energy Expenditure Relationship with Macronutrients and Gestational Weight Gain: A Pilot Study	Nutrients	2,020	cc-by	7,146	Resting Energy Expenditure Relationship with Macronutrients and Gestational Weight Gain: A Pilot Study Kiley B. Vander Wyst Arizona State University Matthew P. Buman Arizona State University Gabriel Q. Shaibi Arizona State University Megan E. Petrov Arizona State University Elizabeth Reifsnider Arizona State University Corrie M Whisner (  cwhisner@asu Arizona State University https://o Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License License:   This work is licensed under a Creative Commons Attribution 4.0 International License. R d F ll Li Page 1/18 Page 1/18 Page 1/18 Abstract Background: Resting energy expenditure (REE) comprises 60% of total energy expenditure and variations may be associated with gestational weight gain (GWG) or maternal diet. The objective of this study was to examine the impact of ambulatory metabolic tracking on GWG and the association with maternal macronutrient composition during mid-pregnancy. Methods: Pregnant women aged 29.8±4.9 yr (78.6% non-Hispanic, White) with gestational ages (GA) < 17 wk were randomized to Breezing™ (N=16) or control (N=12) groups for 13 wks. REE by Breezing™ indirect calorimetry, anthropometrics and dietary intake were collected every two weeks. Early (GA weeks 14-21), late (GA weeks 21-28), and overall (GA week 14-28) changes in macronutrients and GWG were calculated. Results: Overall total and rate of GWG did not differ by group but Breezing™ women had lower rate and total GWG in the beginning of the second trimester but gained significantly more weight [F(1,24)=5.29, p=0.03] in the latter half. Independent of group assignment, early increases in REE were associated with an increased rate of GWG later in pregnancy (r=0.53, p=0.05). Dietary intake did not differ by group at baseline; however, overall energy (-144±339 vs. 192±422, p=0.03) and protein (-10±24 vs. 13±31, p=0.05) intake were significantly lower for the Breezing than the control women which primarily occurred in the latter half of the second trimester. Conclusion: Regular metabolism tracking alone did not have an impact on GWG. Assessment of REE during pregnancy in conjunction with nutritional counseling or education may result in improvements in GWG. Background Overweight and obesity now affect 60% and 35% of reproductive-aged women, respectively.(1) While gestational weight gain (GWG) is a normal part of pregnancy, 47% of women gain more than the recommended amount of weight.(2) Excessive GWG is associated with a multitude of complications that impact both maternal and neonatal health. These adverse short- and long-term health consequences include preeclampsia, gestational diabetes, postpartum weight retention, fetal macrosomia, neonatal hypoglycemia, and admittance to the neonatal intensive care unit.(3,4) Previous research has demonstrated that greater increases in GWG are associated with decreased physical activity, increased energy intake, higher pre-pregnancy BMI, race/ethnicity, and maternal education.(5,6) Recently, research has focused on the evaluation of resting energy expenditure (REE) across pregnancy to better account for the physiological variability in metabolic adaptations to pregnancy that may also impact GWG.(7–11) REE is the amount of energy expended needed to maintain basic organ function, respiration, and circulation which accounts for 60-70% of total daily energy expenditure. Real-time, mobile tracking of REE has become increasingly popular but few devices are able to accurately estimate caloric needs;(12) further, they have rarely been used in pregnant populations.(13,14) Variations in REE during pregnancy have demonstrated an overall increase in caloric needs(8,11,15–17) ranging from 13-35%; however, results remain heterogeneous. Recently, the Breezing™ device, a hand-held, Bluetooth-enabled metabolism tracker, that uses indirect calorimetry to measure REE was developed and validated against the laboratory-based Douglas Bag Method.(18) A previously conducted case series of pregnant women that utilized the Breezing™ device demonstrated improved knowledge and awareness of metabolism, weight Page 2/18 Page 2/18 gain, and caloric intake during pregnancy.(19) This study found unique changes in REE across the participants that were not consistent with predictive equations.(19) It is apparent that REE is a highly variable measure that is impacted by numerous biological (disease status)(20,21) and behavioral (activity level,(9) nutrition,(10) and sleep(22)) factors. Without consideration of REE, dietary and GWG recommendations are based on inaccurate, non-specific predictive equations that may lead to under- or over-nutrition during a critical period of growth and development. Background REE has a strong, positive correlation with total energy, protein, fat, carbohydrates, cholesterol, sugar, and fiber.(10) Total energy intake during pregnancy has been reported to range from approximately 1860 to 2550 kcal/day.(23–27) A recent meta-analysis found that the mean reported intake for total energy, protein, fat, and carbohydrate increased by 184±86 kcal/day, 5.9 gms/day, 10.1 gms/day, and 17.8 gms/day, respectively, from the first to the third trimesters.(27) Although self-reported macronutrient compositions vary among pregnant women,(24–26,28) an estimated 51% of pregnant women have total energy intakes that exceed daily recommendations, with 19% not meeting carbohydrate and 38% exceeding fat recommendations.(29) It is well known that a high quality diet during pregnancy is related to better pregnancy outcomes;(30,31) however, there is a paucity of research investigating not only changes in REE during pregnancy but also how REE is influenced by maternal diet and its impact on rate of weight gain. Obesity during pregnancy has a negative impact on women their offspring. Because more women are entering pregnancy at higher weights, greater efforts are needed to minimize excessive GWG. Assessing and tracking REE throughout pregnancy might provide better insight into factors that impact maternal health, including fluctuations in GWG. The purpose of this study was to evaluate how awareness of REE during the second trimester impacted GWG and how variations in REE were associated with maternal macronutrient consumption. Instruments Demographic & Health Data. Demographic data collected included, occupation, date of birth, age (calculated as current date minus date of birth), marital status, race/ethnicity, and education. A health history questionnaire was completed at each study visit that contained questions about health history and behaviors. Health history items included current gestational age, past medical history, and current use of prescribed or over-the-counter medications and supplements. Health behavior items included current exercise level, dieting status, average per day meal consumption, consumption of caffeinated beverages, average drinks per week for caffeinated beverages, and alcohol consumption. Anthropometric Data. Anthropometrics were collected at every study visit for both groups. Research staff collected height using a portable stadiometer (Seca 213) at the first study visit and weight using a portable scale (Seca 876) at each study visit. These measurements were used to calculate body mass index (BMI) as kg/m2. Total weight gain was determined as the difference between the participant’s weight at Study Visit 7 and Study Visit 1. Study visit 1 occurred during the first trimester where gestational weight gain is minimal. Rate of weight gain was calculated as the total weight gain in kilograms divided by the number of weeks the participant was followed. Resting Energy Expenditure. REE (Breezing™ group only) was determined using the Breezing™ device. REE measurements were obtained every two weeks during the 13-week study to capture variation in metabolic rate. Prior to obtainment of this measurement, participants rested in a seated position for 30 minutes to reduce the impact of recent physical activity on the REE measurement. The participant remained in a seated position when the measurement was being obtained. They were instructed to breathe in and out of the device for two continuous minutes. The Breezing™ device measures oxygen uptake and carbon dioxide production in order to determine REE. Data were loaded onto an accompanying electronic tablet using a corresponding software application and transmitted electronically to the study investigators. If the device indicated irregular breathing, the study participant would perform the measurement for a second time and the average values were used as the final measurement. After the study participant successfully completed the measurement, study staff reviewed the results with them by showing them the data from the Breezing™ device mobile app. The control participants received the same study visits minus use of the Breezing™ device. Study Participants and Procedures This pilot study was a randomized controlled observational trial of pregnant women (n=28). Women were recruited from Obstetrics and Special Supplemental Nutrition Program for Women, Infants, and Children (WIC) at Adelante Healthcare clinics, social media advertising, and word of mouth referral throughout the greater Phoenix metro area (Figure 1) beginning September 2017 through October 2018. Participants were randomized to the Breezing™ (n=16) or the control (n=12) groups and followed for 13 weeks. Device use did not allow for blinding of participants or research staff. Seven home visits were conducted by study staff for data collection. Informed consent and demographic data were obtained at the first in- home study visit. All data were compiled in a secure REDCap database. During the duration of the study, study staff frequently contacted participants by phone or text message to check in, answer any questions or address concerns, and to remind them of upcoming appointments. Page 3/18 Pregnant women with a gestational age <17 weeks and aged >18 years old were eligible to participate. High-risk pregnancies that included the following diseases or conditions were excluded: multiple gestations, fetal growth problems, hypertension, gastrointestinal disorders, malabsorptive diseases, hyper or hypo-parathyroid conditions, HIV, diabetes, asthma/lung disease, cardiac diseases and conditions, current smoker (i.e. women who have smoked 100 cigarettes in their lifetime and now smoke every day or some days), and history of eating disorders. Instruments No dietary or behavioral counseling or medically-relevant support were provided by study staff to either Breezing™ or control group participants. Page 4/18 Dietary Data. Dietary assessments were completed by study staff using the 24-hour dietary recalls(32) at each visit to assess habitual dietary intake during the previous 24-hour period. The 24-hour dietary recall captures detailed information about all foods and beverages consumed by participants. It allows for collection of more detailed information including brands, cooking methods, etc. and probes for commonly forgotten foods, beverages and condiments.(32) Each of the 24-hour dietary recalls were entered into the Nutrition Data System for Research (NDSR) by the same researcher. NDSR is a dietary analysis program designed to collect and analyze data from 24-hour dietary recalls.(33) Statistical Analyses. All statistical procedures were performed using SPSS (SPSS 25, Chicago, IL, USA). Demographic characteristics and baseline factors were summarized using counts and percentages for categorical variables, and the mean and standard deviation or median and interquartile range were reported for continuous measures. Overall rate of GWG was calculated as the overall GWG divided by the total study duration in weeks. Early and late rate of GWG were calculated as the difference in GWG between study visits 4 and 1, and 7 and 4, respectively, divided by the total study duration during each respective time period. Changes in REE, energy and macronutrient (fat, carbohydrates, and protein) consumption were calculated in the same manner. The overall, early, and late changes in rate of and total GWG, and macronutrients were compared between Breezing™ and control groups using a one-way ANCOVA with gestational age at study start and maternal education level as covariates. Adherence to Institute of Medicine rate of GWG recommendations were determined by calculating the number of women who gained the appropriate rate of GWG for second trimester using BMI at the first study visit. BMI at first study visit was used as a proxy for pre-pregnancy BMI since GWG during the first trimester is relatively low. Correlations between the early, late, and overall changes in REE and GWG (total and rate) and macronutrient consumption were estimated using the Pearson Correlation coefficient after controlling for maternal education and initial BMI. All statistical tests were two-sided with significance evaluated at p<0.05. Results There was no difference between groups in total GWG during the first half of the study [F(1,24)=0.38, p=0.54]; however, the Breezing™ group gained significantly more (total GWG) than the Control group during the second half of the study [F(1,24)=5.1, p=0.03]. REE. Data obtained from the Breezing™ device are presented in Table 3. There were non-significant variations in REE throughout the study [F(6,60)=0.14, p=0.99]. 80% (n=12/15) of women had an increase in REE between the second and third study visit (mean GA: 18 to 20 weeks) with increases in REE ranging from 10 to 350 kcal/day. The differences between the remaining study visits were almost evenly split among women who experienced an increase (range: 53-63%) or a decrease (range: 38-50%) in REE. Early changes in REE (72±211 kcals) were relatively small but late changes (128±294 kcals) were nearly twice that of early changes. The mean overall change in REE was 200±316 (range: -340 to 950) kcals. There was an 11.5% increase in total REE between the first and last study visits among the Breezing™ group participants. Association between REE and Rate of GWG. Early changes in REE were not associated with early changes in the rate of GWG (r=-0.26, p=0.33). Similarly, there was no association between late changes in REE and late changes in GWG (r=-0.18, p=0.55). Likewise, there was no relationship between overall changes in REE and overall changes in GWG (r=0.01, p=0.96). However, there was a positive correlation between early changes in REE and late changes in the rate of GWG such that greater increases in REE early in the second trimester were related to greater rate of GWG in the latter half of the second trimester (r=0.54, p=0.03). Macronutrient Composition. There were no statistically significant differences for any of the baseline dietary intake data (Table 4). The early, late, and overall changes in consumption of the macronutrients among the two study groups are provided in Table 5. Overall (mean diff=-349.1±150.8, 95% CI: -660.3 to -37.9, p=0.03) and late (mean diff=-379.9±143.9, 95% CI:-676.9 to -82.9, p=0.01) mean differences in changes in energy consumption were significantly different between the Breezing™ and Control groups. Overall changes in protein were mildly different between Breezing™ and Control group participants (mean diff=-22.5±11.0, 95% CI: -45.2 to 0.3, p=0.05). Likewise, late changes in protein decreased slightly more for the Breezing™ group when compared to the Control group (mean diff=-23.2±11.2, 95% CI: -46.3 to 0.0, p=0.05). Results Demographic and Baseline Data. Of the 34 women who were eligible for the study, 28 were randomized to study groups (16 in the Breezing™ and 12 in the Control groups; Figure 1). All 28 women completed the entire study. The median study duration for the control and Breezing™ groups was 13.4 (12.9, 14.0) and 13.4 (IQR: 13.3, 14.8) weeks, respectively (Kruskal-Wallis H test, χ2=1.2, p=0.27). The mean gestational age was 14.8±2.3 weeks at study visit 1, 17.9±2.7 weeks at study visit 2, 20.2±2.5 weeks at study visit 3, 22.4±2.4 weeks at study visit 4, 24.3±2.7 weeks at study visit 5, 26.7±2.8 weeks at study visit 6, and 28.9±2.6 weeks at study visit 7. Complete demographic data and baseline data are provided in Table 1. Body mass index (BMI) categories of the women at the initial study visit were equally distributed with 35.7% (n=10) classified as normal weight, 35.7% (n=10) as overweight, and 28.6% (n=8) as obese. The distribution across groups by BMI category did not differ (χ2=3.5, p=0.17). Figure 2 provides the distribution of initial study visit BMI category for the two study groups and the entire cohort. Page 5/18 Page 5/18 Intervention Effect on GWG. Table 2 summarizes the rate and total GWG for the study participants. Despite the Breezing™ group having a greater initial weight, both groups experienced similar overall rates of [F(1,24)=-2.0, p=0.17] and total [F(1,24)=1.6, p=0.22] GWG. Compared to the control group, the Breezing™ group had a significantly higher rate of GWG during the second half of the study (F(1,24)=8.15, p=0.01), but not during the early half of the study [F(1,24)=0.56, p=0.46]. There was no difference between groups in total GWG during the first half of the study [F(1,24)=0.38, p=0.54]; however, the Breezing™ group gained significantly more (total GWG) than the Control group during the second half of the study [F(1,24)=5.1, p=0.03]. Intervention Effect on GWG. Table 2 summarizes the rate and total GWG for the study participants. Despite the Breezing™ group having a greater initial weight, both groups experienced similar overall rates of [F(1,24)=-2.0, p=0.17] and total [F(1,24)=1.6, p=0.22] GWG. Compared to the control group, the Breezing™ group had a significantly higher rate of GWG during the second half of the study (F(1,24)=8.15, p=0.01), but not during the early half of the study [F(1,24)=0.56, p=0.46]. Discussion The current pilot study was designed to provide women awareness of actual caloric needs throughout pregnancy and evaluate whether exposure to this information impacted appropriate GWG. The women who used the Breezing™ device gained more weight throughout the 13-week study period when compared to the Control group; however, this difference was not statistically significant. This finding is similar to other studies among pregnant women that have had little to no effect on GWG.(34–38) Surprisingly, there were significant differences in the rate of GWG between study groups depending on study time. In the first half of the study, there was no difference in rate or total GWG between the Breezing™ and Control group participants. This changed in the latter half of the study with the Breezing™ group having a higher total and rate of weight gain. This may indicate that GWG during the second trimester might have initially been impacted by the metabolic tracking but that awareness of REE did not have a lasting impact given that late changes in total and rate of GWG among the Breezing™ group exceeded that of the Control group. Regardless of negligible findings regarding GWG, to our knowledge, this is the first study that has implemented the use of a real-time metabolism tracker among pregnant women to monitor REE and investigate the impact of REE monitoring on GWG. In our study, there was an overall increase in REE throughout the study of 11.5%. This is similar to other studies that have assessed REE that found increases ranging from 13-35%.(8,11,15–17) The smaller increase in the current study could be explained by the shorter evaluation period. Unlike other studies that found a moderate negative correlation between REE in the 3rd trimester and total GWG,(11) we only found a positive correlation between early changes in REE and GWG in the latter half of the study. Our lack of a positive association across the entire study period may be due to the small sample size or large variations in REE at each study visit. Approximately 40% of the women had a decrease in REE which ranged from 10 to 620 kcal/d. Similarly, the women who had an increase in REE had substantial variations ranging from 10 to 350 kcal/d. Results Early changes in protein were marginally increased in the Breezing™ group relative to the Control group (mean diff=20.3±10.2, 95% CI: -0.7 to 41.3, p=0.06). There were no statistically or marginally significant differences for overall, late, and early changes in carbohydrates and fat. Association between REE and Macronutrient Composition. Overall changes in REE were not correlated with overall changes in energy (r=0.24, p=0.38), fat (r=0.25, p=0.34), protein (r=0.36, p=0.17), or Page 6/18 Page 6/18 carbohydrate (r=0.03, p=0.91) consumption. Evaluation of time-specific changes in macronutrients with time-specific changes in REE revealed significant correlations between early changes in REE and overall changes in carbohydrate intake (r=0.58, p=0.02) and a trending significant correlation with overall changes in energy intake (r=0.46, p=0.07). Discussion The current body of work demonstrates the need for a more in- depth evaluation of energy needs during pregnancy and the potential relationship with GWG as some women might experience drastic fluctuations in REE, thereby increasing their risk for excessive GWG or other pregnancy-related health conditions. Future studies may benefit from more regular sampling of REE and monitoring of other behavioral factors (e.g. physical activity) across gestation to fully understand these fluctuations and how they relate to GWG. The current study also assessed maternal diet composition during pregnancy. Baseline intakes of total fat (69 grams/day) for the entire study population were comparable to findings from other studies.(26– 28,39–41) Despite percent fat (35.7%) being slightly above the Acceptable Macronutrient Distribution Range, the mean total energy consumption (1690 kcal/day) among our cohort was much lower than Page 7/18 Page 7/18 Page 7/18 Page 7/18 other studies (1970-2480 kcal/day).(27,28,40,41) Likewise, energy from carbohydrates (48%) was slightly lower (31-62 grams/day less) than previous reports which ranged from 238-269 grams/day.(26– 28,40,41) The percent of calories from protein (17%) was slightly higher than other studies which have reported a range from 14-16%.(26,27) However, total daily protein (68.3 grams) consumption among the current study participants was lower than the national average (78.1 grams) for pregnant women.(27) Despite these minor differences, the overall macronutrient composition of the current cohort of pregnant women resembles that of other studies. When assessing early, late, and overall changes in other studies (1970-2480 kcal/day).(27,28,40,41) Likewise, energy from carbohydrates (48%) was slightly lower (31-62 grams/day less) than previous reports which ranged from 238-269 grams/day.(26– 28,40,41) The percent of calories from protein (17%) was slightly higher than other studies which have reported a range from 14-16%.(26,27) However, total daily protein (68.3 grams) consumption among the current study participants was lower than the national average (78.1 grams) for pregnant women.(27) Despite these minor differences, the overall macronutrient composition of the current cohort of pregnant women resembles that of other studies. When assessing early, late, and overall changes in macronutrients, we observed an overall increase in energy and all macronutrients for the Control group whereas the Breezing™ group had an overall decrease; however, time-specific changes varied between groups. Discussion Previous studies have found inconsistent findings pertaining to changes in macronutrient intake throughout pregnancy.(39,42) A previous meta-analysis of 90 studies (n=126,242) demonstrated an increase of 184±86 kcal, 5.9 grams of protein, 10.1 grams of fat, and 17.8 grams of carbohydrates per day from the first to the third trimester.(39) The current study demonstrates the variation in maternal diet across pregnancy and the need to have better dietary assessment methods in order to capture accurate changes. There are several strengths of the current study including the randomized controlled design and overall study adherence. Furthermore, the longitudinal collection of data allowed for evaluation of changes, providing a more complete picture of the fluctuations among these factors during pregnancy. The current study is not without limitations. A major limitation of this study was the small sample size which might have impacted our ability to find significant results. Furthermore, our sample consisted of primarily White, highly educated women which might not be generalizable to the broader population. Lastly, the time of REE measurement collection of may have been inaccurate. REE should be measured immediately upon waking prior to consumption of any food or before any exercise. However, this was not possible as the study visits were scheduled at the most convenient times for the study participants. In order to allow for this flexible but to adjust for potential inaccuracies from measuring REE at variable times, all participants were asked to sit at rest for a minimum of thirty minutes prior to obtaining REE measures. Abbreviations ANCOVA – analysis of covariance BMI – body mass index GWG – gestational weight gain NDSR - Nutrition Data System for Research SPSS – Statistical Package for Social Sciences REE – resting energy expenditure Conclusions Although both groups experienced similar overall and rate of GWG, the Breezing™ group had significantly greater total and rate of GWG during the second half of the study compared to the Control group. Further, the Breezing™ group had an increase in the overall and late changes in total energy expenditure and protein whereas the Control group had a decrease. This also study demonstrated substantial fluctuations in REE, with an approximate 11.5% increase, indicating the need to account for individualized physiological needs during pregnancy and evaluation of their impact on GWG. Early changes in REE correlated with late changes in the rate of GWG demonstrating that early shifts in energy requirements might impact GWG later in pregnancy. Next steps include exploring these relationships with a larger population during the entire course of a pregnancy and the feasibility of integration of the Breezing™ device into healthcare providers’ offices to be used during well-woman and prenatal visits to stimulate discussion about nutritional needs and obtain REE information from preconception through postpartum. Page 8/18 Page 8/18 Consent for publication: Not applicable Consent for publication: Not applicable Availability of data and materials: The datasets used and analyzed in the current study are available from the corresponding author on reasonable request. Declarations Ethics approval and consent to participate: Written informed consent was obtained from all participants. Recruitment of participants began after Arizona State University Institutional Review Board approval was obtained. The date of approval was Competing Interests: None Funding: Project HoneyBee (PHB) at Arizona State University provides funding for researchers that are investigating the utility of commercial devices used for monitoring various health parameters. PHB has no role in the design of the study, the collection, analysis, and interpretation of data nor in writing the manuscript. Author Contributions: KBV, ER, and CMW conceptualized and designed the study, obtained funding, coordinated and supervised data collection, and critically reviewed the manuscript for important intellectual content. MPB, GQS and MEP critically reviewed the manuscript for important intellectual content. Page 9/18 Page 9/18 MPB supervised the initial analyses. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. MPB supervised the initial analyses. All authors approved the final manuscript as submitted and agree to be accountable for all aspects of the work. Acknowledgments: We would like to thank the women in this study for volunteering to participate and for welcoming us into their homes. Additionally, we would like to thank the research assistants who helped with participant recruitment and data collection. References 1. Center for Disease Control and Prevention. Table 58. Normal weight, overweight, and obesity among adults aged 20 and over, by selected characteristics: United States, selected years 1988-1994 through 2013-2016. Health (Irvine Calif) [Internet]. 2017;1–9. Available from: https://www.cdc.gov/nchs/hus/contents2017.htm#054 1. Center for Disease Control and Prevention. Table 58. Normal weight, overweight, and obesity among adults aged 20 and over, by selected characteristics: United States, selected years 1988-1994 through 2013-2016. Health (Irvine Calif) [Internet]. 2017;1–9. Available from: https://www.cdc.gov/nchs/hus/contents2017.htm#054 2. The American College of Obstetricians and Gynecologists. ACOG Committee Opinion. 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Participant Demographics and Baseline Anthropometrics, Behavioral, and Prenatal Data Total (n=28) Control (n=12) Breezing™ (n=16) P-value Maternal age, mean±SD 29.8±4.9 29.6±5.9 29.9±4.3 0.86 0.36 ---- ---- ---- ---- 0.71 ---- ---- ---- ---- ---- 0.62 ---- ---- ---- 0.17 ---- ---- ---- ---- ---- Race/Ethnicity, % (n) Non-Hispanic Caucasian 78.6 (22) 75.0 (9) 81.3 (13) Hispanic Caucasian 14.3 (4) 25.0 (3) 6.25 (1) Asian 3.6 (1) 0 (0) 6.25 (1) More than one race 3.6 (1) 0 (0) 6.25 (1) Employment classification, % (n) Service and sales workers 14.3 (4) 16.7 (2) 12.5 (2) Managerial jobs 3.6 (1) 0 (0) 6.3 (1) Professional 39.3 (11) 50.0 (6) 31.3 (5) Clerical support workers 7.1 (2) 8.3 (1) 6.3 (1) Stay at home mom 35.7 (10) 25.0 (3) 43.8 (7) Marital Status, % (n) Single 3.6 (1) 0 (0) 6.3 (1) Married 85.7 (24) 91.7 (11) 81.3 (13) Partnered/Significant Other 10.7 (3) 8.3 (1) 12.5 (2) Education Level, % (n) Less than 8th grade 3.6 (1) 0 (0) 6.3 (1) High School/GED 10.7 (3) 25.0 (3) 0 (0) Two-year college 17.9 (5) 8.3 (1) 25.0 (4) Four-year college 32.1 (9) 25.0 (3) 37.5 (6) Post-graduate 35.7 (10) 41.7 (5) 31.3 (5) Weight (kgs), mean±SD 73.7±16.0 70.3±15.8 76.2±16.2 0.35 Height (cms), mean±SD 164.1.5 163.5±6.6 164.4±6.6 0.72 Body Mass Index (kg/m2), mean±SD 27.4±5.8 26.3±5.8 28.2±5.9 0.40 Gestational age (weeks), mean±SD 14.8±2.2 15.3±1.5 14.4±2.7 0.29 Exercise Level, % (n) 0.32 Sedentary 14.3 (4) 25.0 (3) 6.3 (1) ------ Mild 57.1 (16) 50.0 (6) 63.5 (10) ------ Occasional vigorous 21.4 (6) 25.0 (3) 18.8 (3) ------ Regular vigorous 7 1 (2) 0 (0) 12 5 (2) ------ Prenatal Vitamin Use, % (n) 82.1 (23) 83.3 (10) 81.3 (13) 0.89 Employment categories were determined using the International Standard Classification of Occupations-08. Exercise level were defined as the following: sedentary (no exercise), mild (climbing stairs, waking a few blocks, golfing), occasional vigorous (less than 4 times per week for 30 mins), and regular vigorous (at least 4 times per week for 30 mins or more). Medication use included both prescribed and over-the-counter medicines. Prenatal vitamin use was based off self-report and did not include multivitamin use. Independent t- tests were performed for all continuous variables to determine if there were statistically significant differences between groups and mean (SD) are reported. Tables Chi-Square tests were evaluated for all baseline categorical variables to determine if there were statistically significant difference between groups. Abbreviations: SD= standard deviation; Table 3. Mean±SD of Resting Energy Expenditure from Breezing™ Device Resting Energy Expenditure Study Visit 1 1544±237 Study Visit 2 1511±220 Study Visit 3 1628±242 Study Visit 4 1616±253 Study Visit 5 1708±338 Study Visit 6 1706±367 Study Visit 7 1744±384 Comparisons between timepoints were assessed using repeated measures analysis of variance with covariates of maternal education level and gestational age at study start. All data are presented as mean±SD and were obtained from the Breezing™ device. Abbreviations: SD=standard deviation. 0.89 Table 2. Mean±SD of Rate of and Total Gestational Weight Gain (GWG) Among Study Participants Control (n=12) Breezing™ (n=16) Total (n=28) Cohen’s d P-Value Rate of GWG Overall Changes 0.5±0.3 0.6±0.2 0.5±0.2 0.4 0.17 Early Changes 0.6±0.3 0.5±0.3 0.5±0.3 0.4 0.46 Late Changes 0.4±0.3 0.7±0.3 0.6±0.3 1.1 0.01 Total GWG Overall Changes 7.1±2.6 8.1±2.9 7.7±2.8 0.4 0.22 Early Changes 4.2±1.7 3.6±2.0 3.9±1.9 0.3 0.54 Late Changes 3.0±1.4 4.4±1.7 3.8±1.7 0.9 0.03 ANCOVA with maternal education and gestational at study start were performed to compare group differences in rate and total GWG and mean (SD) were reported. Cohen’s d was calculated using means and SDs. Cohen’s d= .2 is a small effect, =.5 is a moderate effect, =.8 is a large effect. Abbreviations: SD= standard deviation; GWG= gestational weight gain; IQR= interquartile range, defined as the difference between the third and first quartile. Table 3. Mean±SD of Resting Energy Expenditure from Breezing™ Device Resting Energy Expenditure Study Visit 1 1544±237 Study Visit 2 1511±220 Study Visit 3 1628±242 Study Visit 4 1616±253 Study Visit 5 1708±338 Study Visit 6 1706±367 Study Visit 7 1744±384 Comparisons between timepoints were assessed using repeated measures analysis of variance with covariates of maternal education level and gestational age at study start. All data are presented as mean±SD and were obtained from the Breezing™ device. Abbreviations: SD=standard deviation. Page 15/18 Page 15/18 Page 15/18 Table 4. Baseline Maternal Macronutrient, Fiber, Cholesterol, Caffeine, and Sugar Consumption at the Initial Study Visit Total (n=28) Control (n=12) Breezing™ (n=16) P-Value Total Energy (kcal) 1690.2±495.7 1751.1±493.8 1644.6±508.2 0.58 Total Fat (gm) 68.8±29.3 71.0±29.0 67.2±30.3 0.75 Saturated Fat (gm)a 22.2 (13.1, 33.4) 18.8 (11.3, 56.6) 26.1 (7.8, 40.7) 1.00 Monounsaturated Fat (gm) 56.7±25.3 27.3±14.3 23.8±12.2 0.49 Polyunsaturated Fat (gm) 28.5±13.0 12.5±5.7 13.4±8.0 0.73 Cholesterol (mg)a 178.9 (94.5, 283.3) 185.2 (89.4, 273.5) 166.1 (103.3, 292.0) 0.85 Total Carbohydrate (gm) 207.8±58.2 219.2±55.9 199.2±60.3 0.38 Total Dietary Fiber (gm) 19.6±9.6 22.1±11.2 17.7±8.1 0.23 Soluble Fiber (gm) 15.3±5.9 6.6±3.5 5.4±2.3 0.29 Insoluble Fiber (gm) 13.6±7.5 15.4±8.5 12.3±6.7 0.28 Total Protein (gm) 68.3±24.0 68.6±24.0 68.0±24.7 0.95 Caffeine (mg)a 3.1 (0.0, 47.4) 2.9 (0.0, 88.5) 3.5 (0.0, 44.5) 0.69 Total Sugars (gm)a 69.6 (51.7, 107.2) 78.7 (66.0, 104.5) 62.3 (42.9, 121.1) 0.33 Added Sugars (gm) 38.9±29.4 32.0±20.7 44.0±34.4 0.26 Independent samples t-tests were performed for all normally distributed data and mean (SD) were reported. 0.89 a Kruskal-Wallis non-parametric test was performed for all non-normally distributed data and median (IQR) were reported. Abbreviations: SD= standard deviation; IQR= interquartile range, defined as the difference between the third and first quartile; kcal=kilocalories; mg=milligrams; gm=grams. All data are based on an interview conducted 24-hour dietary recall at the initial study visit. Independent samples t-tests were performed for all normally distributed data and mean (SD) were reported. a Kruskal-Wallis non-parametric test was performed for all non-normally distributed data and median (IQR) were reported. Abbreviations: SD= standard deviation; IQR= interquartile range, defined as the difference between the third and first quartile; kcal=kilocalories; mg=milligrams; gm=grams. All data are based on an interview conducted 24-hour dietary recall at the initial study visit. Independent samples t-tests were performed for all normally distributed data and mean (SD) were reported. a Kruskal-Wallis non-parametric test was performed for all non-normally distributed data and median (IQR) were reported. Abbreviations: SD= standard deviation; IQR= interquartile range, defined as the difference between the third and first quartile; kcal=kilocalories; mg=milligrams; gm=grams. All data are based on an interview conducted 24-hour dietary recall at the initial study visit. Independent samples t-tests were performed for all normally distributed data and mean (SD) were reported. a Kruskal-Wallis non-parametric test was performed for all non-normally distributed data and median (IQR) were reported. Abbreviations: SD= standard deviation; IQR= interquartile range, defined as the difference between the third and first quartile; kcal=kilocalories; mg=milligrams; gm=grams. All data are based on an interview conducted 24-hour dietary recall at the initial study visit. Table 5. Mean±SD for Overall, Late, and Early Changes by Group (N=28) Variable Control (n=12) Breezing™ (n=16) Cohen’s d P-Value Energy (kcal) Overall Changes 191.8±422.2 -143.8±339.2 0.18 0.03 Early Changes -43.0±766.1 32.2±642.6 0.00 0.79 Late Changes 207.1±416.5 -155.3±309.22 0.23 0.01 Protein (gm) Overall Changes 13.3±31.0 -9.9±24.0 0.15 0.05 Early Changes -9.9±19.9 7.4±30.8 0.14 0.06 Late Changes 13.4±33.0 -10.0±22.9 0.15 0.05 Carbohydrates (gm) Overall Changes 19.3±57.8 -14.5±58.7 0.10 0.12 Early Changes -2.4±73.6 1.8±87.9 0.00 0.86 Late Changes 19.3±58.0 -14.4±55.8 0.11 0.11 Fat (gm) Overall Changes 7.4±25.5 -5.5±14.9 0.10 0.11 Early Changes 0.5±60.8 -0.4±28.0 0.00 0.87 Late Changes 7.8±25.2 -5.8±14.5 0.11 0.09 ANCOVA was performed with maternal education and gestational age at study start as covariates to test for group differences. Abbreviations: SD= standard deviation; kcal=kilocalories; gm=grams. group differences. Abbreviations: SD= standard deviation; kcal=kilocalories; gm=grams. Figures Page 16/18 gure 1 Figure 1 Page 17/18 Figure 2 Page 18/18 Page 18/18
https://openalex.org/W2123455734	https://ijponline.biomedcentral.com/counter/pdf/10.1186/1824-7288-40-S2-A4	English	null	Rooming-in organization to prevent neonatal mortality and morbidity in late preterm infants	The Italian Journal of Pediatrics/Italian journal of pediatrics	2,014	cc-by	949	Open Access Open Access Table 1 Assessment and care of the late preterm infant [3]. Assess gestational age of neonate Assess and monitor respiratory status Appropriate respiratory interventions Assess for risk factors and symptoms of heat loss and/or cold stress Interventions to maintain a neutral thermal environment Interventions and assessment of hypoglycemia including transfer to higher acuity unit or facility if indicated Assess for maternal and neonatal risk factors for sepsis Antibiotic therapy and diagnostic evaluation if sepsis is suspected Assess for presence of jaundice and hyperbilirubinemia Phototherapy as indicated y Parent education regarding signs and symptoms of jaundice and hyperbilirubinemia Breastfeeding, and support for breastfeeding mothers including observation, education and validation Discharge planning including parent education, counseling, and validation of knowledge about recognizing and acting on risk factors Parent education regarding signs and symptoms of jaundice and hyperbilirubinemia Breastfeeding, and support for breastfeeding mothers including observation, education and valid Discharge planning including parent education, counseling, and validation of knowledge about r 1Pediatric Unit, Maternal and Child Health Department, S. Giacomo Hospital, ASL BA, Monopoli (Bari), Italy. 2Neonatology and Neonatal Intensive Care Unit, Department of Biomedical Science and Human Oncology, University of Bari “Aldo Moro”, Bari, Italy. Rooming-in organization to prevent neonatal mortality and morbidity in late preterm infants Mariano Manzionna1, Antonio Di Mauro2 Mariano Manzionna1, Antonio Di Mauro2 From XX National Congress of the Italian Society of Neonatology Rome, Italy. 9-11 October 2014 Evidence of physiologic maturity, feeding competency, thermoregulation and absence of medical of medical ill- ness are minimum discharge criteria for late-preterm newborns. Furthermore it’s of great importance to assess educational programs with special instruction and gui- dance to parents, engaging families in providing appro- priate home care after hospital discharge. A long term follow-up arrangements is also recommended to assess and plan early interventions in case of neurodevelop- ment delay [4]. Despite most infants born at 34+0 through 36+7 weeks’ gestation are thought to be at low risk during the birth hospitalization and have a neonatal course with no sig- nificant complications, they are physiologically and metabolically immature with an higher rates of morbid- ity and mortality than term infants [1]. Most common medical condition associated with late- preterm births are respiratory distress, apnea, tempera- ture instability, hypoglycemia, hypocalcemia, jaundice, poor feeding, sepsis and finally an higher rates of the hospital readmissions during the neonatal period. These morbidities result in workup for sepsis evaluations, anti- biotic therapy, intravenous fluid administration, ventila- tory support and increased length of stay with higher hospital costs [2]. We conclude that, based on the significant morbidity and mortality of late preterm births, the health care focus on prematurity should be expanded to include the late preterm period. Rooming-in organization of late preterms births aims to assess and identify risk factors, prevent and manage potential medical complications during hospitalization. Interventions and practices reccomended are illustred in table 1. Manzionna and Di Mauro Italian Journal of Pediatrics 2014, 40(Suppl 2):A4 http://www.ijponline.net/content/40/S2/A4 Manzionna and Di Mauro Italian Journal of Pediatrics 2014, 40(Suppl 2):A4 http://www.ijponline.net/content/40/S2/A4 * Correspondence: mariano.manzionna@alice.it 1Pediatric Unit, Maternal and Child Health Department, S. Giacomo Hospital, ASL BA, Monopoli (Bari), Italy Full list of author information is available at the end of the article © 2014 Manzionna and Di Mauro; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 2 Page 2 of 2 Manzionna and Di Mauro Italian Journal of Pediatrics 2014, 40(Suppl 2):A4 http://www.ijponline.net/content/40/S2/A4 Published: 9 October 2014 References 1. Engle WA, Tomashek KM, Wallman C: “Late-Preterm” Infants: A Population at Risk. Pediatrics 2007, 120(6):1390-1401. 2. Engle WA: Infants Born Late Preterm: Definition, Physiologic and Metabolic Immaturity, and Outcomes. Neoreviews 2009, 10(6):280-286. 3. Association of Women’s Health, Obstetric, and Neonatal Nurses (AWHONN): Assessment and Care of the Late Preterm Infant Evidence-Based Clinical Practice Guidelines. Washington, DC: AWHONN; 2010. 4. Kugelman A, Colin AA: Late preterm infants: Near Term But Still in a Critical Developmental Time Period. Pediatrics 2013, 132(4):741-751. doi:10.1186/1824-7288-40-S2-A4 Cite this article as: Manzionna and Di Mauro: Rooming-in organization to prevent neonatal mortality and morbidity in late preterm infants. Italian Journal of Pediatrics 2014 40(Suppl 2):A4. References 1. Engle WA, Tomashek KM, Wallman C: “Late-Preterm” Infants: A Population at Risk. Pediatrics 2007, 120(6):1390-1401. 2. Engle WA: Infants Born Late Preterm: Definition, Physiologic and Metabolic Immaturity, and Outcomes. Neoreviews 2009, 10(6):280-286. 3. Association of Women’s Health, Obstetric, and Neonatal Nurses (AWHONN): Assessment and Care of the Late Preterm Infant Evidence-Based Clinical Practice Guidelines. Washington, DC: AWHONN; 2010. 4. Kugelman A, Colin AA: Late preterm infants: Near Term But Still in a Critical Developmental Time Period. Pediatrics 2013, 132(4):741-751. doi:10.1186/1824-7288-40-S2-A4 Cite this article as: Manzionna and Di Mauro: Rooming-in organization to prevent neonatal mortality and morbidity in late preterm infants. Italian Journal of Pediatrics 2014 40(Suppl 2):A4. References doi:10.1186/1824-7288-40-S2-A4 Cite this article as: Manzionna and Di Mauro: Rooming-in organization to prevent neonatal mortality and morbidity in late preterm infants. Italian Journal of Pediatrics 2014 40(Suppl 2):A4. doi:10.1186/1824-7288-40-S2-A4 Cite this article as: Manzionna and Di Mauro: Rooming-in organization to prevent neonatal mortality and morbidity in late preterm infants. Italian Journal of Pediatrics 2014 40(Suppl 2):A4. 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https://openalex.org/W3179130068	https://repository.ubn.ru.nl/bitstream/handle/2066/235507/1/235507.pdf	English	null	Towards OPtimal TIming and Method for promoting sUstained adherence to lifestyle and body weight recommendations in postMenopausal breast cancer survivors (the OPTIMUM-study): protocol for a longitudinal mixed-method study	BMC women's health	2,021	cc-by	11,632	Towards OPtimal TIming and Method for promoting sUstained adherence to lifestyle and body weight recommendations in postMenopausal breast cancer survivors (the OPTIMUM-study): protocol for a longitudinal mixed-method study Cappellen-van Maldegem, Sandra J.M. Van; Mols, Floortje; Horevoorts, Nicole; Kruif, Anja de; Buffart, L.M.; Schoormans, Dounya; Seidell, Jacob C.; Hoedjes, Meeke 2021, Article / Letter to editor (BMC Women's Health, 21, 1, (2021), article 268) Doi link to publisher: https://doi.org/10.1186/s12905-021-01406-1 Note: To cite this publication please use the final published version (if applicable). van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 https://doi.org/10.1186/s12905-021-01406-1 Towards OPtimal TIming and Method for promoting sUstained adherence to lifestyle and body weight recommendations in postMenopausal breast cancer survivors (the OPTIMUM‑study): protocol for a longitudinal mixed‑method study Sandra J. M. van Cappellen‑van Maldegem1, Floortje Mols1,2, Nicole Horevoorts1,2, Anja de Kruif3,9, Laurien M. Buffart4, Dounya Schoormans1, Hester Trompetter1, Sandra Beijer2, Nicole P. M. Ezendam1,2, Michiel de Boer5,6, Renate Winkels7, Ellen Kampman7, Jantine Schuit1, Lonneke van de Poll‑Franse1,2,8, Jacob C. Seidell5 and Meeke Hoedjes1* the OPTIMUM research team © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background: The majority of postmenopausal breast cancer (PMBC) survivors do not adhere to lifestyle recommen‑ dations and have excess body weight. In this group, this is associated with poorer health-related quality of life and an increased risk of type II diabetes mellitus, cardiovascular disease, second primary cancers, cancer recurrences, and mortality. Gaining and maintaining a healthy lifestyle and body composition is therefore important. It is unknown when and how sustained adherence to these recommendations can be promoted optimally in PMBC survivors. Therefore, the OPTIMUM study aims to identify the optimal timing and method for promoting sustained adherence to lifestyle and body weight recommendations in PMBC survivors. Methods: The OPTIMUM-study has a mixed-methods design. To assess optimal timing, a longitudinal observational study will be conducted among approximately 1000 PMBC survivors. The primary outcomes are adherence to lifestyle and body weight recommendations, readiness for change, and need for support. Questionnaires will be adminis‑ tered at 4–6 months after cancer diagnosis (wave 1: during treatment and retrospectively before diagnosis), 1 year after diagnosis (wave 2: after completion of initial treatment), and 1.5 years after diagnosis (wave 3: during follow-up). Wave 2 and 3 include blood sampling, and either wearing an accelerometer for 7 days or completing a 3-day online food diary (randomly assigned at hospital level). To assess the optimal method, behavioural determinants of the primary outcomes will be matched with Behavior Change Techniques using the Behaviour Change Technique Tax‑ onomy. Qualitative research methods will be used to explore perceptions, needs and preferences of PMBC survivors Correspondence: m.hoedjes@tilburguniversity.edu 1 CoRPS ‑ Center of Research On Psychological and Somatic Disorders, Department of Medical and Clinical Psychology, Tilburg University, PO Box 90153, 5000 LE Tilburg, the Netherlands Full list of author information is available at the end of the article Correspondence: m.hoedjes@tilburguniversity.edu 1 CoRPS ‑ Center of Research On Psychological and Somatic Disorders, Department of Medical and Clinical Psychology, Tilburg University, PO Box 90153, 5000 LE Tilburg, the Netherlands Full list of author information is available at the end of the article Correspondence: m.hoedjes@tilburguniversity.edu 1 CoRPS ‑ Center of Research On Psychological and Somatic Disorders, Department of Medical and Clinical Psychology, Tilburg University, PO Box 90153, 5000 LE Tilburg, the Netherlands Full list of author information is available at the end of the article Background generally ready to change their lifestyle whereas non-par- ticipants are not. Ideally, adherence should also be pro- moted in those not ready for lifestyle change. A large body of evidence has demonstrated that higher levels of body fatness, adult excessive weight gain, drink- ing alcohol, and physical inactivity increase the risk of postmenopausal breast cancer (PMBC) [1, 2]. PMBC survivors are defined as people who are living with a diagnosis of PMBC, including those who have recovered from the disease [1]. PMBC survivors with an unfavora- ble lifestyle and body composition have a lower health- related quality of life (HRQoL), an increased risk for type II diabetes mellitus, cardiovascular disease, second pri- mary cancers, cancer recurrences, and mortality [3–6]. Several biological mechanisms, such as enhanced inflam- mation, underlie these health-related outcomes [7–9]. To increase HRQoL and decrease the risk of the devel- opment of comorbidities and mortality [10–15], lifestyle and body weight recommendations have been issued [1, 16]. However, the majority of PMBC survivors does not meet these recommendations [1, 17–22]. For this reason, readiness for lifestyle change should be taken into account in promoting lifestyle, since each stage of change ((not ready: pre-contemplation/con- templation); (ready: preparation/action/maintenance); (relapse: relapse) [29]) requires different behavior change techniques [29–31]. Oncology health-care professionals play a key role in lifestyle-related information provision to cancer survivors. (Oncology) health care professionals may promote readiness for lifestyle change, since receiv- ing a cancer diagnosis has been marked as a ‘teachable moment’ to promote adherence [32]. Unfortunately, life- style and body weight recommendations for cancer sur- vivors are currently not well imbedded in Dutch health care. Although oncology health-care professionals play a key role in information provision to cancer survivors, they do not routinely provide information on the health benefits of meeting lifestyle and body weight recommen- dations (e.g., lower risk of all-cause, cancer-specific, and cardiovascular disease morbidity and mortality [33]). Although numerous studies have shown that lifestyle interventions result in, mostly short-term, improvements in lifestyle and body weight in cancer survivors, the opti- mal timing and method to enhance long-term adherence to lifestyle and body weight recommendations remains unknown [23, 24]. Previous studies have used a top-down approach to promote adherence to recommendations in cancer survivors. These studies have generally applied (adapted versions of) interventions that have previously been proven effective in other populations. © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 2 of 14 (semi-structured interviews, focus groups) and health care providers (Delphi study). Topics include perceptions on optimal timing to promote adherence; facilitators and motivators of, and barriers towards (sustained) adherence to recommendations; and acceptability of the selected methods. Discussion: The OPTIMUM study aims to gain scientific knowledge on when and how to promote sustained adher‑ ence to lifestyle and body weight recommendations among PBMC survivors. This knowledge can be incorporated into guidelines for tailored promotion in clinical practice to improve health outcomes. Keywords: Postmenopausal breast cancer survivors, Body weight, Lifestyle, Stages of change, Need for support, Behavior Change Techniques, Mixed-method design, Patient reported outcomes, Biomarkers, PROFILES registry Background So far, this approach has not led to increased insight into the opti- mal method and timing to promote sustained adherence to recommendations in cancer survivors. Accumulation of scientific evidence is hindered by several factors. For instance, poor reporting of intervention components in the scientific literature [25, 26], and a lack of extensive process evaluations to identify effective intervention components and underlying behavior change mecha- nisms. Moreover, intervention studies are typically not designed to assess optimal timing of lifestyle support. In addition, these studies typically promote adherence to recommendations in those who are ready to change their lifestyle [27, 28], as intervention participants are In addition, a different approach of lifestyle support by (oncology) health care professionals is required for those with and without a perceived need for support for improving or maintaining a (healthy) lifestyle. For those who perceive a need for support, receiving infor- mation is not sufficient to achieve adherence, and addi- tional support should be offered [34]. Such support should be tailored to one’s needs and preferences to promote uptake of, compliance to, and effectiveness of support [34]. Tailoring promotion of adherence to indi- vidual characteristics, is in line with current consensus on the importance of personalized care [35]. Such tai- loring typically does not incorporate the variety of con- sequences of cancer and its treatment that may act as barriers or facilitators for lifestyle change after a cancer diagnosis. For example, impaired psychological health (e.g. depressive symptoms) is typically not taken into account while promoting lifestyle change in cancer sur- vivors. However, impaired psychological health is rela- tively common up to years after a cancer diagnosis [36] van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 3 of 14 1d. To explore perceptions on optimal timing among PMBC survivors, oncology health care professionals, and other relevant stakeholders. and negatively related to health behaviors (e.g., being physically inactive) [37, 38]. A more holistic approach to promoting health behavior change includes incor- poration of traditional health behavior change deter- minants (e.g., self-efficacy) as well as the barriers and facilitators related to physical and psychological health after cancer diagnosis and treatment. Key objective 2: To gain insight into the optimal method for (oncology) health care professionals to promote (sus- tained) adherence to lifestyle and body weight recom- mendations in subgroups of PMBC survivors. Background In contrast with the top-down approach to promo- tion of health behavior change in cancer survivors used in previous studies, the OPTIMUM-study will use a bot- tom-up approach (i.e., building scientific evidence from basic psychosocial research, rather than from applica- tion of existing complex interventions) for individualised intervention development from knowledge on specific modifiable determinants relevant for PMBC survivors. By matching specific modifiable determinants relevant for this specific patient population to behavior change techniques, a ‘toolbox’ containing a variety of building blocks (i.e., intervention ingredients) can be composed. This toolbox can be used to create individualized inter- ventions by selecting the right tools for each specific individual. g This is further specified into the following sub-objectives: 2a. To compose ‘patient profiles’ according to ‘adher- ence to a particular recommendation’, ‘readiness for change’, and ‘need for support’; 2a. To compose ‘patient profiles’ according to ‘adher- ence to a particular recommendation’, ‘readiness for change’, and ‘need for support’; • To describe which patient profiles are most preva- lent per time point; • To describe socio-demographic and clinical char- acteristics of the most frequent patient profiles. 2b. To assess personal, clinical, and cancer-related modifiable determinants of adherence, readiness for change, and need for support in PMBC survivors over time; To accumulate scientific evidence on the optimal tim- ing and method to promote sustained adherence to lifestyle and bodyweight recommendations in PMBC survivors, the OPTIMUM-study (Towards OPtimal TIm- ing and Method for promoting sUstained adherence to lifestyle and body weight recommendations in post- Menopausal breast cancer survivors) was initiated. The OPTIMUM-study uses a systematic, bottom-up, holis- tic approach [39]. The overall aim is to gain insight into the optimal timing and method to promote (sustained) adherence to lifestyle and bodyweight recommendations in (subgroups) of PMBC survivors.h • To gain knowledge on which determinants should be targeted to promote sustained adherence;i • To describe modifiable determinants of the most frequent patient profiles. 2c. To select Behavior Change Techniques [30] that could be used to influence the associated modifi- able determinants (i.e., toolbox containing potential intervention ingredients). The OPTIMUM study has two key objectives with sev- eral sub-objectives: 2d. To explore the acceptability of the selected Behav- ior Change Techniques, and to explore perceptions on the optimal method to promote (sustained) adher- ence among PMBC survivors, oncology health care professionals, and other relevant stakeholders. Background Key objective 1: To gain insight into the optimal timing to promote (sustained) adherence to lifestyle and body weight recommendations in PMBC survivors. This is further specified into the following sub-objectives: Design and methods Design 1a. To longitudinally assess proportions of PMBC survivors’ non-adherence and need for support to be able to improve lifestyle or maintain lifestyle improvements. 1a. To longitudinally assess proportions of PMBC survivors’ non-adherence and need for support to be able to improve lifestyle or maintain lifestyle improvements. 1a. To longitudinally assess proportions of PMBC survivors’ non-adherence and need for support to be able to improve lifestyle or maintain lifestyle improvements. 1b. To examine socio-demographic and clinical char- acteristics of those who do (not) adhere and of those who (not) indicate a need for support over time. 1c. To examine biological markers in relation to life- style and bodyweight of those who do (not) adhere and of those who (not) indicate a need for support over time. Design To increase knowledge on optimal timing for promotion of sustained adherence in PMBC survivors, the OPTI- MUM-study longitudinally assesses adherence to lifestyle and body weight recommendations, readiness for change, and need for support to be able to adhere to these rec- ommendations over time. To increase knowledge on the optimal method for promotion of sustained adherence in PMBC survivors, modifiable determinants of health behavior change relevant for this specific patient popu- lation will be assessed and matched to behavior change 1b. To examine socio-demographic and clinical char- acteristics of those who do (not) adhere and of those who (not) indicate a need for support over time. 1c. To examine biological markers in relation to life- style and bodyweight of those who do (not) adhere and of those who (not) indicate a need for support over time. van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 4 of 14 techniques. That knowledge will be used to obtain a ‘toolbox’ of ‘building blocks’ (i.e. behavior changes tech- niques) that can be used in composing an individually tailored intervention for PMBC patients [30]. PMBC sur- vivors will be categorized into ‘patient profiles’ accord- ing to the answers to the following questions: (1) Does one need to change their lifestyle behavior to be able to adhere to a particular lifestyle or body weight recommen- dation? (as assessed by adherence to a particular lifestyle recommendation), (2) To what extent is one ready to change her lifestyle behavior? (assessed by readiness for change), and (3) Is one able to achieve change by herself or does she need support to be able to improve a specific health behavior? (assessed by need for support). Each patient profile requires a different combination of behav- iour changes techniques (building blocks) to promote health behaviour change. See “Appendix” for an overview of patient profiles. Qualitative measurements will include semi-structured interviews based on purposive sampling at wave 2 and wave 3, focus groups after the interviews, and a Delphi- study. The qualitative research methods will be used to explore perceptions, needs and preferences of PMBC survivors (semi-structured interviews, focus groups) and health care providers (Delphi study). See Fig. 1 for an overview of the design of the OPTIMUM-study. Study population l Inclusion criteria are having been diagnosed with breast cancer 4 to 6 months ago and being postmenopausal (i.e., not having menstruated for at least 1 year). Exclusion cri- teria are having been diagnosed with a Ductal Carcinoma in Situ and not being able to independently understand and complete a Dutch questionnaire, or being inter- viewed in Dutch. i The OPTIMUM-study is a longitudinal observational study with a mixed-methods design, comprising both quantitative and qualitative measurements. Quantitative measurements will include questionnaires at 4–6 months after cancer diagnosis (wave 1: during treatment, with retrospective measurement before diagnosis), 1 year after diagnosis (wave 2: after completion of initial treatment), and 1.5 years after diagnosis (wave 3: during follow-up). As additional markers of adherence, at wave 2 and 3 quantitative measurements will include blood sampling (in 9 out of 16 participating hospitals) and either wear- ing an accelerometer for 7 days, or completing an online 3-day food diary (randomly assigned at hospital level). Recruitment ll Patients will be invited for study participation by their own oncology health-care professional (i.e., oncolo- gist, internist, surgeon, or mamma care nurse) from 16 participating hospitals across the Netherlands. Eligible patients will receive an invitation letter during a visit to their oncology health care professional. After providing written informed consent, participants will be invited to complete either an online or paper version of the first questionnaire (wave 1). According to their preference for completing either an online or paper version of the ques- tionnaire, participants will be contacted for data collec- tion at waves 2 and 3. Participants who prefer to complete Fig. 1 Design of the OPTIMUM-study. van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 5 of 14 spent on paid or unpaid work, and household activities [43]. The PASE has shown to have good to excellent test–retest reliability, and to be a rea- sonably valid method to classify healthy elderly individuals and cancer patients into categories of physical activity [43–45]. a paper version of the questionnaire will receive printed questionnaires by mail. Participants who prefer to com- plete an online version of the questionnaire will receive a link to the online questionnaire via e-mail. Online ques- tionnaires will be completed via the PROFILES (Patient Reported Outcomes Following Initial treatment and Long term Evaluation of Survivorship) registry [27]. In case of non-response, one reminder will be sent per par- ticipant per wave (either via e-mail or by mail according to their preference), followed by up to 5 telephone calls. The OPTIMUM study aims to recruit approximately 1000 participants. Approximately 25 PMBC survivors will be invited for semi-structured interviews based on purposive sampling according to (non)adherence and need for support over time as assessed by means of ques- tionnaires in wave 2 and 3. p y y [ ] • Detailed data on participants’ physical activity and sedentary behaviours will be collected using an accelerometer, the ActiGraph wGT3X [46]. Survivors treated in a hospital selected for wear- ing the accelerometer will wear an accelerometer on their wrist for 7 consecutive days on their non- dominant arm. Upon return of the ActiGraph, the data will be downloaded using the accompany- ing software ActiLife (Version 6.13.3; ActiGraph, Pensacola, FL, USA) and saved in raw format. Subsequently, the.gt3x files are converted to time- stamp free.csv files which could be exported into R v.3.6.0. Quantitative measures • Five-Times-Sit-to-Stand (FTSTS) test): this test will be used to determine lower body muscle func- tion, and may indicate sarcopenia and frailty [50]. Participants will perform this test at home using a chair and a stopwatch (included in the informa- tion package). Participants will measure the time it takes to stand up and sit down five times from a chair. This test has been found valid and reliable to assess lower body muscle function [50]. a. Overweight and body fat distribution. Excess body weight and body fat distribution will be determined by self-reported height and weight with which we cal- culate BMI [15] and self-measured hip- and waist cir- cumference [40]. The waist circumference and waist/ hip ratio provides an indication of body fat distribu- tion (i.e. abdominal fat accumulation) and associated disease risk [41]. b. Physical activity and sedentary behaviour Measurements Table 1 provides an overview of all quantitative measures at wave 1, wave 2, and wave 3. Table 2 provides an overview of the study criteria used to determine (non-)adherence to the lifestyle and body weight recommendations of the World Cancer Research Fund (WCRF) [1, 2], as well as the recommendation for sleep of the American Academy of Sleep Medicine and Sleep Research Society (AASM&SRS) [16]. Recruitment ll The.csv files are processed using the R-package GGIR v.2.1-0 [47, 48]. Data of partici- pants will be excluded from subsequent analysis if their accelerometer files demonstrated a post- calibration error bigger than 0.01 g; if there are less than 3 valid wear-days (defined as ≥ 16 h per day) [49]; or if there are no wear data present for each 15 min period of the 24 h cycle. Physical activity level will be expressed as average accelera- tion across the day (Eucledian Norm Minus One (ENMO), mg) [49], intensity gradient across the day (IG), average time accumulated in low inten- sity physical activity (LPA) per day (min/day), average time accumulated in moderate-to-vigor- ous physical activity (MVPA) per day (min/day), average time accumulated in vigorous physical activity (VPA) per day (min/day), time spent inac- tively per day (min/day), and most active continu- ous 30 min (M30) per day. Participants are not informed about lifestyle and body weight recommendations as part of the OPTIMUM study because of its observational nature. As such, whether or not participants are informed about the recommenda- tions depends on the standard care they receive. Standard care for participants currently does not include informa- tion provision about lifestyle and body weight recom- mendations, although differences between hospitals and health care professionals do exist. c. Dietary intake • Physical activity will be assessed with the Physi- cal Activity Scale for the Elderly (PASE) [42], a 13-item questionnaire that assesses participation in leisure activities. In addition, muscle strength- ening activities will be recorded, as well as time • Food diary: Dietary intake will be assessed by ask- ing the patients to complete an online food diary (the ‘Eetmeter’, a digital tool of the Netherlands Nutrition Center) to register all foods and drinks, • Food diary: Dietary intake will be assessed by ask- ing the patients to complete an online food diary (the ‘Eetmeter’, a digital tool of the Netherlands Nutrition Center) to register all foods and drinks, van Cappellen‑van Maldegem et al. Table 1 (continued) Variables Table 1 (continued) Table 1 (continued) Variables Instrument Wave 1a Wave 2a Wave 3a Objective Mental and physical fatigue Multidimensional Fatigue Inventory (MFI) [65] x x 2b, 2c Symptoms of depression and anxiety Hospital anxiety and depression scale (HADS) [66] x x 2b, 2c Biological determinants of cancer prognosis Inflammation Pro- and anti-inflammatory cytokines (TNFα, IL-6, IL-10, IL-1Ra) and CRP x x 1c Metabolism leptin, insulin, insulin growth factor-1, glucose, HbA1C, total cholesterol, triglycerides, HDL cholesterol, LDL cholesterol, Vitamin D x x 1c a wave 1 = 4–6 months after diagnosis; wave 2 = 1 year after diagnosis; wave 3 = 1.5 years after diagnosis a wave 1 = 4–6 months after diagnosis; wave 2 = 1 year after diagnosis; wave 3 = 1.5 years after diagnosis Table 2 Overview of study measures to determine (non-)adherence to the lifestyle and body weight recommendations of the World Cancer Research Fund (WCRF) [1, 2], as well as the recommendation for sleep of the American Academy of Sleep Medicine and Sleep Research Society (AASM&SRS) [16] Lifestyle and bodyweight recommendations Operationalization of recommendation Measurement instrument used to assess recommendation Weight [1] BMI between 18.5 and 24.9 kg/m2Waist circumference below 80 cm Standardized questions weight and height Self-administered hip- and waist circumference measurement Physical activity [1] At least 150 min of low intensity exercise during 1 week, spread over several days At least 2 times a week muscle and bone strengthening exercises Prevent sitting too much and limit sedentary behavior Questionnaire: The Physical Activity Scale for the Elderly (PASE) [42] Actigraph (7 days): average acceleration (AvAcc), intensity gra‑ dient (IG), total minutes light, moderate and vigorous physi‑ cal activity per day, total minutes of inactive time per day, most active continuous 30 min (M30) per day.(randomized at hospital level) [46] Wholegrains, veg‑ etables, fruit and beans [1] Eat at least 250 g of vegetables each day Eat at least 2 pieces of fruit each day Eat beans at least once a week Eat at least 30 g of wholegrains each day Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary (randomized at hospital level) Fast foods [1] Limit consumption of processed foods high in fat, starches or sugar—including fast foods: any pre-prepared dishes, snacks, bakery foods, deserts, and confectionary (candy) Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary (randomized at hospital level) Meat products [1] Eat no more than 350 to 500 g of red or processed meat per week Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary (randomized at hospital level) Sugary drinks [1] Drink mostly water and unsweetened drinks Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary randomized at hospital level) Alcoholic drinks [1] Drink no alcohol Standardized questions alcohol consumption Smoking [2] Do not smoke Standardized smoking questions Sleep [16] Sleep at least 7 h per night Questionnaire: Pittsburg Sleep Quality Index [55] Actigraph (7 days): sleep latency, sleep efficiency, daytime sleep, frequency of long sleep interruptions (> 5 min), total minutes of sleep per night (randomized at hospital level) [46] Table 2 Overview of study measures to determine (non-)adherence to the lifestyle and body weight recommendations of the World Cancer Research Fund (WCRF) [1, 2], as well as the recommendation for sleep of the American Academy of Sleep Medicine and Sleep Research Society (AASM&SRS) [16] Measurement instrument used to assess recommendation Standardized questions weight and height Self-administered hip- and waist circumference measurement Questionnaire: The Physical Activity Scale for the Elderly (PASE) [42] Actigraph (7 days): average acceleration (AvAcc), intensity gra‑ dient (IG), total minutes light, moderate and vigorous physi‑ cal activity per day, total minutes of inactive time per day, most active continuous 30 min (M30) per day.(randomized at hospital level) [46] Standardized questions weight and height S lf d i i t d hi d i t i f Standardized questions weight and height Self-administered hip- and waist circumferen Wholegrains, veg‑ etables, fruit and beans [1] Eat at least 250 g of vegetables each day Eat at least 2 pieces of fruit each day Eat beans at least once a week Eat at least 30 g of wholegrains each day Fast foods [1] Limit consumption of processed foods high in fat, starches or sugar—including fast foods: any pre-prepared dishes, snacks, bakery foods, deserts, and confectionary (candy) Meat products [1] Eat no more than 350 to 500 g of red or processed meat per week Sugary drinks [1] Drink mostly water and unsweetened drinks Alcoholic drinks [1] Drink no alcohol Smoking [2] Do not smoke Sleep [16] Sleep at least 7 h per night Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary (randomized at hospital level) Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary (randomized at hospital level) Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary (randomized at hospital level) Questionnaire: Dutch Healthy Eating Index [54] Online 3 day Food diary randomized at hospital level) Standardized questions alcohol consumption Standardized smoking questions Questionnaire: Pittsburg Sleep Quality Index [55] Actigraph (7 days): sleep latency, sleep efficiency, daytime sleep, frequency of long sleep interruptions (> 5 min), total minutes of sleep per night (randomized at hospital level) [46] tein, and carbohydrate) consumption automati- cally. c. Dietary intake BMC Women’s Health (2021) 21:268 van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 7 of 14 c. Dietary intake BMC Women’s Health (2021) 21:268 Page 6 of 14 Table 1 Overview of quantitative measures and measurement instruments in the OPTIMUM-study Variables Instrument Wave 1a Wave 2a Wave 3a Objective Sociodemographic and health-related variables Demographics (education, marital status, employment status) Demographic questions x x x 1b Comorbidities Self-administered Comorbidity Questionnaire (SCQ) [60] x x x 1b Estrogen/breast cancer related variables Questionnaire items concerning age of onset menarche in years, number of pregnancies, total duration of breastfeeding, age of onset menopause in years x 1b Cancer-specific health related quality of life European Organization for Research and Treat‑ ment Quality of Life Questionnaire (EORTC QLQ-C30) [61] x x 1a Overweight and body fat distribution BMI Weight in kg/(Height in m)2 x x x 1a, 2a Hip- and waist circumference Self-administrated measurement by use of meas‑ urement tape x x x 1a, 2a Physical activity + sedentary behavior Physical activity level and sedentary behavior Physical Activity Scale for the Elderly (PASE) [42] x x x 1a, 2a Physical activity accelerometry 7-day accelerometer data (ActiGraph) [46] Physical activity measures: Average acceleration (AvAcc), Intensity Gradient (IG), total minutes light, moderate and vigorous physical activity per day, total minutes of inactive time per day, most active continuous 30 min (M30) per day x x 1a, 2a Functional muscle strength 5Times-Sit-To-Stand functional muscle strength measurement: self-administrated measurement by use of stopwatch [50] x x 2b Dietary intake Diet quality (including alcohol consumption) Dutch Healthy Diet—index 15 (DHD-15), with minor adjustments [54] x (shortened) x x 1a, 2a Dietary intake: energy and macronutrients Online 3-day food diary: registration of all foods and drinks, in portion sizes of gram/ml, they have consumed during the day using the ‘Eet‑ meter’ from the Dutch ‘Voedingscentrum’ x x 1a, 2a Smoking Smoking behaviours Smoking behaviour questions x x x 1a, 2a Sleep Sleep quality and disturbances Pittsburgh Sleep Quality Index (PSQI) [55] x x 1a, 2a Sleep accelerometry 7-day accelerometer data (wristworn ActiGraph wGT3X) [46] Sleep measures: sleep latency, sleep efficiency, day‑ time sleep, frequency of long sleep interruptions (> 5 min), total minutes of sleep per night x x 1a, 2a Lifestyle and health related measures Readiness for lifestyle change Assessed according to the transtheoretical model (not ready: pre-contemplation/contempla‑ tion); (ready: preparation/action/maintenance); (relapse: relapse) [29] with 1 item per recom‑ mendation x x x 2a, 2b, 2c Need for support Need for support assessed with 1 item per recom‑ mendation x x x 1a, 2a, 2b, 2c Posttraumatic growth Posttraumatic Growth Inventory (PGI) [62] x x 2b, 2c Self-compassion Short Form Self-Compassion Scale [63]: 6 positive items only x x x 2b, 2c Emotion regulation Cognitive Emotion Regulation Questionnaire (CERQ)—short [64] x x 2b, 2c ble 1 Overview of quantitative measures and measurement instruments in the OPTIMUM-study van Cappellen‑van Maldegem et al. g. Biological markers in relation to lifestyle and body- weight. g. Biological markers in relation to lifestyle and body- weight. Blood will be collected by venipuncture at the par- ticipants’ treating hospital. All participants, in 9 out of 16 participating hospitals, will receive a lab form in their information package (at wave 2 and wave 3). Attached to this lab form, they will receive a short questionnaire. This questionnaire contains questions concerning fasted state, medication use, and sickness at the moment of blood sampling, as these factors can have an impact on the biological markers of interest. Time of blood donation will be marked on the ques- tionnaire. Directly after blood sampling, the serum blood sample will be allowed to clot at room temper- ature and will be centrifuged. The EDTA blood sam- ple will be centrifuged at room temperature directly after blood sampling. The subtracted plasma, serum, and buffy coat samples will be processed within 2 h of collection and are stored at − 80 °C until further analyses. All procedures will be defined in a protocol to ensure standardisation over study sites. All blood samples will be transported from the laboratory at the treating hospitals to the Biobank Maastricht. Fol- lowing, appropriate ELISAs and ILLUMINA analyses will take place to determine the biological markers. Blood samples will be stored in a biobank for later analysis of biomarkers. Analysis of the following bio- markers is anticipated: • Detailed data on participants’ sleep pattern will be collected by use of an accelerometer, the Acti- Graph wGT3X [46]. Participants will wear the accelerometer during the night (in total 7 nights) to obtain data on: sleep duration, sleep latency, wake after sleep onset, sleep interruptions, and sleep efficiency. d. Smoking • Smoking will be assessed by standardized ques- tions on smoking habits (i.e., cigarettes/shag, cigars, pipe tobacco, and e-cigarettes). PMBC survivors will be classified in; never, ex, light, and heavy smokers. • Perceived need for support. At all measurement points and for each specific lifestyle and body weight recommendation, participants will be asked if they are in need for support to be able to change their lifestyle and/or body weight. Also, they will be asked to specify the type of support they would prefer by use of an open-ended ques- tion. e. Sleep e. Sleep • Sleep quality and disturbances will be measured using the Pittsburgh Sleep Quality Index (PSQI) [55] which assesses sleep quality and disturbances over a one-month period. Nineteen items measure seven ‘component’ scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep effi- ciency, sleep disturbances, use of sleeping medica- tion, and daytime dysfunction. The sum of these seven component scores add up to one global score. The total global component score ranges from 0 to 21. Higher scores indicate lower sleep quality and more sleep disturbances [55]. The PSQI is known for its good validity, it is able to discriminate good from poor sleepers. In addition, internal homogeneity and consistency (test–retest reliability) are good. Table 1 (continued) Variables in portion sizes or gram/ml, they have consumed during the day [51]. At wave 2 and wave 3, patients will be asked to register their daily intake during three days: two week days and one weekend day. The Eetmeter is connected to the Dutch Food Composition Database (NEVO) [52, 53] which allows for the calculation of the quantity of daily energy, micro-, and macronutrients (i.e., fat, pro- • Adherence to dietary guidelines: Diet quality will be assessed by use of the Dutch Healthy Diet index-15 (DHD-15) [54]. The DHD-15 is a brief food frequency questionnaire that estimates diet quality and assesses adherence to the fifteen food-based Dutch dietary guidelines of 2015 van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 8 of 14 ready: pre-contemplation/contemplation); (ready: preparation/action/maintenance); (relapse: relapse) [29] and will be assessed for each recom- mendation with a single item. (e.g., fruit, vegetables, wholegrain products, leg- umes, nuts, diary, fish, tea, fats and oils, coffee, red and processed meat, sweetened beverages and fruit juices, alcohol, and salt). Per compo- nent, the scores range from 0 to 10, resulting in a total score between 0 (no adherence) to 150 (complete adherence). The ability of the DHD-15 to rank persons on their diet quality is consid- ered to be acceptable [54]. Several of the Dutch Dietary Guidelines are similar to the WCRF recommendations, therefore, the results of the DHD-15 will also provide insight into adherence to the WCRF recommendations. For each of the lifestyle recommendations, par- ticipants will be asked to indicate which stage of change fits their current state or their state just before diagnosis (i.e., wave 1) best with self- designed questions (see Table 1). If patients have attempted to change but could not maintain this change, they automatically relapse to a prior stage of the transtheoretical model. For this reason they will be allowed to tick boxes of two stages of change, both ‘relapse’ and either ‘precontempla- tion’, ‘contemplation’, or ‘preparation’ [29, 31]. d. Smoking Quantitative data Descriptive statistics and Generalized Linear Mixed Models (GLMM) will be used to: (1a) longitudinally assess proportions of (non-)adherence to each recom- mendation, readiness for change, and the need for sup- port, to (1b) examine sociodemographic and clinical characteristics, and to (1c) examine biological determi- nants, of those who do not adhere and of those in need for support over time. The relation between adherence to each recommendation and socio-demographic and non- changeable clinical characteristics will be longitudinally assessed by fitting GLMM with adherence to each recom- mendation (no/yes) as dependent dichotomous variable and time (wave1, wave2, wave3) and socio-demographic and clinical characteristics (age, ethnicity, socioeconomic status, marital status, stage of cancer at diagnosis, type of treatment) as independent variables. We will assess the need to include interaction terms between time and the socio-demographic and clinical characteristics. These analyses will be repeated for need for support as outcome variable. Similar analyses will be conducted for biological determinants of cancer prognosis modifiable by lifestyle and bodyweight. h. Clinical cancer-related variables h. Clinical cancer-related variables Data on clinical cancer-related variables will be retrieved from the Netherlands Cancer Registry (NCR), which records clinical data of all newly diag- nosed cancer patients in the Netherlands. h. Clinical cancer-related variables h. Clinical cancer-related variables f. Lifestyle and health related measures For each single lifestyle recommendation, the follow- ing possible changeable determinants of adherence will be determined. • Readiness for lifestyle change will be measured according to the transtheoretical model (not van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 9 of 14 • Inflammation. Pro- and anti-inflammatory cytokines will be determined, including Tumor Necrosis Factor-alpha (TNFα), Interleukin-6 (IL-6), Interleukin-10 (IL-10), and Interleukin-1 Receptor Antagonist (IL-1Ra), and a general marker of inflammation C-reactive protein (CRP). from the observer will be supplemented to the tran- scripts. Results of each focus group will be discussed between the moderator and the observer. c. Delphi-study c. Delphi-study An iterative three-round online Delphi study will be conducted to gain insight in perceptions of medical health care professionals (i.e., mamma oncology sur- geons, mamma oncology internal medics, mamma oncology nurses, oncology dieticians, oncology phys- ical therapists, oncology psychologists), policy mak- ers, and PMBC survivors of potential barriers and facilitators for promoting lifestyle adherence in daily clinical practice. The three rounds will be respec- tively used for item generation, prioritizing of items, and ranking of the items. l • Metabolism: biomarkers include leptin, insulin, insulin growth factor-1, glucose, glycated haema- globin (HbA1C1), total cholesterol, triglycerides, High-Density-Lipoprotein (HDL) cholesterol, Low-Density-Lipoprotein (LDL) cholesterol, and vitamin D. a. Interviews Semi-structured interviews will be held to explore perceptions on optimal timing for support (Key objective 1) and to gain insight in possible change- able determinants of adherence to lifestyle and body- weight recommendations. PMBC survivors will be invited for semi-structured interviews based on pur- posive sampling according to (non)adherence, readi- ness for change, and need for support over time as assessed by means of questionnaires in wave 2 and 3. The number of invited participants depends on the information that comes up during the interviews. Interviews will be guided by a topic list. Discussion topics include barriers, facilitators, and motivators for adherence to recommendations in daily clinical practice, and perceptions on optimal timing of pro- motion of adherence. Interviews will be audiotaped and transcribed verbatim. Transcripts from the inter- views will be supplemented with field notes from the interviewer. Member checking will be performed after the interviews (i.e. returning a summary of an interview to a participant to check for accuracy and whether it resonated with their experiences) [56]. With regard to aim 2a, for each single lifestyle recom- mendation ‘patient profiles’ will be composed by creating a cross tabulation of the variables ‘adherence to a particu- lar recommendation’ (yes/no), ‘readiness for change’ (Not ready: pre-contemplation/contemplation; ready: prepara- tion/action/maintenance), and ‘need for support’ (yes/no) for each time point (see “Appendix”). Based on these cross tabulations, it will be examined which patient profiles are most prevalent per time point. In addition, descriptive statistics will be used to describe changeable socio-demo- graphic (e.g., employment, education) and clinical charac- teristics (e.g., tumor stage, treatment received) of the most frequent patient profiles. GLMM will be used to assess b. Focus groups Focus groups will be conducted after the interviews to validate and enrich the data gathered during the interviews, to prioritize possible changeable deter- minants of adherence, and to further explore themes that arise during the interviews. Focus groups will be audiotaped and transcribed verbatim. Field notes van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 10 of 14 timing and method to enhance lifestyle in PMBC survi- vors (key objective 1 and 2). timing and method to enhance lifestyle in PMBC survi- vors (key objective 1 and 2). a. Interviews socio-demographic and clinical modifiable determinants of adherence, readiness for change, and need for support, as captured by the most prevalent patient profiles over time (aim 2b). We will assess the need to include interac- tion terms between time and the socio-demographic and clinical characteristics. Based on the behavior change technique taxonomy [30], the changeable socio-demo- graphic and clinical determinants will be matched to suit- able behaviour change techniques (aim 2c). Additionally, with respect to aim 2a and 2b, the composition of ‘patient profiles’, multilevel latent class modelling will be used com- bining adherence to all recommendations, readiness for change to each specific recommendation, and need for support for each specific recommendation, for each time point for all recommendations. The multilevel latent class model will be used to gain insight into the course of the patient profiles over time. Stakeholder group A stakeholder group will be actively involved throughout the study, in order to provide a solid basis for implemen- tation and dissemination of study findings. This group consists of 10–15 stakeholders, including representatives of: the Dutch breast cancer patient association, profes- sional bodies for health care professionals such as oncol- ogists, oncology nurses, and general practitioners; policy makers, and a representative of health insurance compa- nies. Stakeholders will be: informed about study progress, consulted for advice on issues that may arise throughout the study, involved in decision making, and in writing a plan for adoption, implementation, sustainability, and evaluation of guidelines on how and when to promote Qualitative data h b Research objectives 1d and 2d, will be addressed by means of qualitative analysis. Specifically, exploring per- ceptions on optimal timing and method among PMBC survivors, oncology health care professionals, and other relevant stakeholders. With respect to the interviews and focus groups, a thematic analysis will be conducted as described in Braun and Clarke [57]. Transcripts will be subsequently disentangled, divided into fragments and open-coded. Codes will be categorized by subthemes and main themes. Relationships between the subthemes will be explored, to eventually cover the subthemes under the overall themes. The codes, subthemes, and themes will be discussed by two researchers until consensus is reached. Codes and (sub)themes will be structured in a code tree. The constant comparison method will be used in order to understand the differences, as well as similarities, between respondents and within each of the respond- ents. The main results will be discussed in the research team to enhance the robustness of the findings. Furthermore, to be able to detect valid associations between (non-)changeable socio-demographic and clinical characteristics and the most prevalent patient profiles per time point (aim 2a and 2b), power analy- sis indicated a minimum of at least 1076 participants. Power analysis was based on an ANCOVA including 5 groups (expected number of main patient profiles in the cross-tabulation based on ‘adherence to a particular rec- ommendation’, ‘readiness for change’, and ‘need for sup- port’) and 3 covariates (e.g., stage of cancer at diagnosis), assuming a small effect for each predictor (partial eta squared = ηp 2 = 0.015). i The output of the rounds of the Delphi-study (aim 1d and 2d) will be analysed (i.e., defining items, categorizing items, removal of duplicate items, calculating sum scores for prioritizing and ranking of items). Thereafter, the out- put will be used as input for the next round till, in con- sultation with the oncology medical professionals, a top rank of facilitators and barriers for lifestyle care will be created in the third round. Sample sizeh The sample size calculation was conducted using the validated rule of thumb of a minimum of 10 participants per independent variable in the smallest group of the dichotomous outcome measure (e.g., 25% non-adherence [20, 58] vs. 75% adherence to the recommendation on alcohol intake) [59]. For aim 2b, incorporating the high- est number of changeable determinants, a maximum of 16 changeable determinants will be incorporated in the analyses. Based on data on adherence to recommenda- tions from previous studies in Dutch cancer survivors [20, 58], the largest number of participants needed to be able to detect valid associations between changeable determinants and adherence to each recommendation with inclusion of 16 independent variables is 860 for the recommendation for smoking (assuming 18.6% of women smoke) (1610)/18.6 × 100). The required number of participants for the other recommendations are: 462 (160/34.62 × 100) for body weight; 601 (160/26.62 × 100) for physical activity; 375 (160/42.62 × 100) for foods and drinks that promote weight gain; 351 (160/45.6 × 100) for fruit intake; 580 (160/27.6 × 100) for vegetable intake; and 624 (160/25.66 × 100) for alcohol intake. Combined data Stage of change [29] Adherence to a particular WCRF- recommendation Does not meet recommendation Meets recommendation Precontem- plation (not ready): not intending to take action in the next six months No need for support Cancer survivors who do not meet a WCRF-recom‑ mendation, do not intend to change their behavior, and do not perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, do not intend to change their behavior, and do not perceive a need for support Need for sup‑ port Cancer survivors who do not meet a WCRF-recom‑ mendation, do not intend to change their behavior, and perceive a need for supporta Cancer survivors who meet a WCRF-recom‑ mendation, do not intend to change their behavior, and report a per‑ ceived need for supporta Contempla- tion (get‑ ting ready): intending to take action in the next 6 months No need for support Cancer survivors who do not meet a WCRF-rec‑ ommendation, intend to change in the foresee‑ able future, and do not perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, intend to change in the foresee‑ able future, and do not perceive a need for support Need for sup‑ port Cancer survivors who do not meet a WCRF-rec‑ ommendation, intend to change in the foresee‑ able future, and perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, intend to change in the foresee‑ able future, and perceive a need for support Ready for change Preparation (ready): ready to take action in the next 30 days No need for support Cancer survivors who do not meet a WCRF-rec‑ ommendation, intend to take action in the immediate future, and do not perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, intend to take action in the immediate future, and do not perceive a need for support Need for sup‑ port Cancer survivors who do not meet a WCRF-rec‑ ommendation, intend to take action in the immediate future, and perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, intend to take action in the immediate future, and perceive a need for support Discussion In most PMBC patients lifestyle and bodyweight are suboptimal [1, 17–22], which may be related to unhealthy lifestyle behaviors. The OPTIMUM-study aims to provide scientific evidence on when and how to promote sustained adherence and in which PMBC patients. The study leads to products (i.e. a toolbox) that can be used in clinical practice to promote sus- tained adherence to lifestyle and bodyweight recom- mendations in PMBC patients. Data security/disclosure of original documentsi Confidentiality and anonymity of participants will be guaranteed by assigning a study number to each partic- ipant. All collected data will all be stored in a secured location for 15 years. Ethical considerationsh The study protocol has been reviewed and approved by the medical research ethics committee METC Bra- bant (Medical Research Ethics Committee Brabant, the Netherlands, reference number: NL66913.028.18). In addition, the study has been reviewed and approved by the local ethics committees of the participating centers. Combined data Quantitative results obtained from the measurements and questionnaires will be combined with the qualitative results obtained from the individual interviews and focus group sessions. Together, these data sets will provide a more complete and comprehensive evaluation of optimal van Cappellen‑van Maldegem et al. BMC Women’s Health (2021) 21:268 Page 11 of 14 adherence. In addition, stakeholders will be consulted individually by telephone or e-mail when necessary. adherence. In addition, stakeholders will be consulted individually by telephone or e-mail when necessary. Funding The study is supported with a personal grant from the Dutch Cancer Society (project nr 10960) awarded to MH, and an Investment Subsidy Large (#91101002) of the Netherlands Organization for Scientific Research (The Hague, The Netherlands). These funding bodies did not have any role in the design of this study, and do not have any role in the collection, analyses and interpretation of data and in writing any of the manuscripts that will result from this study. Cancer survivors who meet a WCRF-recom‑ mendation, have been maintain‑ ing lifestyle changes for at least 6 months, and who do not perceive a need for support Availability of data and materials After finishing the data collection, the data will be freely available for non- commercial scientific research, subject to study question, privacy and confi‑ dentiality restrictions, and registration (www.profilesregistry.nl). Cancer survivors who meet a WCRF-recom‑ mendation, have been maintain‑ ing lifestyle changes for at least 6 months, and who do not perceive a need for support to maintain their changes Trial statush of Life Questionnaire; AvAcc: Average acceleration; IG: Intensity Gradient; M30: Most active continuous 30 min per day; FTSTS: Five-Times-Sit-To-Stand; NEVO: Dutch Nutrients Database; TNFα: Tumour Necrosis Factor alpha; IL-6: Interleukin-6; IL-10: Interleukin-10; IL-1Ra: Interleukin-1 Receptor Antagonist; CRP: C-reactive protein; HbA1C1: Glycated haemaglobin; HDL-cholesterol: High-Density-Lipoprotein cholesterol; LDL-cholesterol: Low-Density-Lipopro‑ tein cholesterol; DHEAS: Dehydroepiandrosterone sulfate; NCR: Netherlands Cancer Registry; GDPR: General Data Protection Regulation; IKNL: Netherlands Comprehensive Cancer Organisation. Authors’ contributions MH designed the study, in collaboration with FM and JS. All authors contrib‑ uted to the development of the study protocol. SC, MH and FM drafted the manuscript. All authors read and approved the final manuscript. Competing interests The authors declare that they have no competing interests. Acknowledgements We would like to thank all oncologists, nurses, and research coordinators involved with the OPTIMUM-study in the following hospitals and institutions for their cooperation: Alexander Monro Hospital, Bilthoven; Albert Schweitzer Hospital, Dordrecht; Amphia Hospital, Breda; Canisius Wilhelmina Hospital, Nijmegen; Diakonessenhuis, Utrecht; Elisabeth—Twee Steden Hospital, Tilburg; Jeroen Bosch Hospital, Den Bosch; Medical Centre Leeuwarden, Leeuwarden; Medical Spectrum Twente, Enschede; Renier de Graaf Hospital, Delft; Sint Antonius Hospital, Utrecht; Sint Jansdal Hospital, Harderwijk; VieCuri Medical Centre, Venlo. Ethics approval and consent to participate The study protocol has been reviewed and approved by the medical research ethics committee (Medical Research Ethics Committee Brabant, the Nether‑ lands, reference number: NL66913.028.18). In addition, the study has been reviewed and approved by the local ethics committees of the participat‑ ing centers. All participants will provide written informed consent prior to participation. Trial statush BMC Women’s Health (2021) 21:268 Page 12 of 14 Page 12 of 14 Stage of change [29] Adherence to a particular WCRF- recommendation Does not meet recommendation Meets recommendation Action: has made overt lifestyle changes in the past 6 months No need for support Cancer survivors who do not meet a WCRF-recom‑ mendation, but have made overt lifestyle changes in the past 6 months, and do not perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, have made overt lifestyle changes in the past 6 months, and do not perceive a need for support Need for sup‑ port Cancer survivors who do not meet a WCRF-recom‑ mendation, but have made overt lifestyle changes in the past 6 months, and who perceive a need for support (to maintain life‑ style changes) Cancer survivors who meet a WCRF-recom‑ mendation, have made overt lifestyle changes in the past 6 months, and who perceive a need for support (to maintain life‑ style changes) Maintenance: doing a new behav‑ ior for more than six months No need for support Cancer survivors who do not meet a WCRF-recom‑ mendation, but have been main‑ taining lifestyle changes for at least 6 months, and who do not perceive a need for support Cancer survivors who meet a WCRF-recom‑ mendation, have been maintain‑ ing lifestyle changes for at least 6 months, and who do not perceive a need for support Need for sup‑ port Cancer survivors who do not meet a WCRF-rec‑ ommendation, but have been maintaining lifestyle changes for at least 6 months, and who perceive a need for support to maintain their changes Cancer survivors who meet a WCRF-recom‑ mendation, have been maintain‑ ing lifestyle changes for at least 6 months, and who do not perceive a need for support to maintain their changes aU lik l i t d ll f f of Life Questionnaire; AvAcc: Average acceleration; IG: Intensity Gradient; M30: Most active continuous 30 min per day; FTSTS: Five-Times-Sit-To-Stand; NEVO: Dutch Nutrients Database; TNFα: Tumour Necrosis Factor alpha; IL-6: Interleukin-6; IL-10: Interleukin-10; IL-1Ra: Interleukin-1 Receptor Antagonist; CRP: C-reactive protein; HbA1C1: Glycated haemaglobin; HDL-cholesterol: High-Density-Lipoprotein cholesterol; LDL-cholesterol: Low-Density-Lipopro‑ tein cholesterol; DHEAS: Dehydroepiandrosterone sulfate; NCR: Netherlands Cancer Registry; GDPR: General Data Protection Regulation; IKNL: Netherlands Comprehensive Cancer Organisation. Trial statush The inclusion of patients started in February 2019. Patients will be followed up for 1.5 years after diagno- sis. The COVID-19 pandemic has delayed the inclusion of PMBC survivors in the OPTIMUM-study. van Cappellen‑van Maldegem et al. aUnlikely scenario, expected cell-frequency of near zero. References Havel PJ, Kasim-Karakas S, Mueller W, Johnson PR, Gingerich RL, Stern JS. Relationship of plasma leptin to plasma insulin and adiposity in normal weight and overweight women: effects of dietary fat content and sus‑ tained weight loss. J Clin Endocrinol Metab. 1996;81(12):4406–13. 28. Abdel-Qadir H, Austin PC, Lee DS, et al. A population-based study of cardiovascular mortality following early-stage breast cancer. JAMA Cardiol. 2017;2(1):88–93. tained weight loss. J Clin Endocrinol Metab. 1996;81(12):4406–13. 8. Irwin ML, Varma K, Alvarez-Reeves M, et al. 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Abbreviations 1 CoRPS ‑ Center of Research On Psychological and Somatic Disorders, Depart‑ ment of Medical and Clinical Psychology, Tilburg University, PO Box 90153, 5000 LE Tilburg, the Netherlands. 2 Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht, the Netherlands. 3 Department of Epidemiology and Biostatistics, Amsterdam Public Health (APH), VUmc, Amsterdam, the Netherlands. 4 Radboudumc, Department of Physiology, Radboud Institute for Health Sciences, Nijmegen, the Netherlands. 5 Department of Health Sciences and the Amsterdam Public Health Research Institute, VU University Amsterdam, Amsterdam, the Netherlands. 6 Department of General Practice and Elderly Care Medicine, UMCG, Groningen, the Netherlands. 7 Division of Human Nutrition and Health, Wageningen University, Wageningen, the Netherlands. 8 Department of Psychosocial Research and Epidemiology, Netherlands Cancer Institute, Amsterdam, the Netherlands. 9 Department OPTIMUM: Towards OPtimal TIming and Method for promoting sUstained adherence to lifestyle and bodyweight recommendations in postMenopau‑ sal breast cancer survivors; PMBC: Postmenopausal breast cancer; HRQoL: Health-related quality of life; PROFILES: Patient Reported Outcomes Following Initial treatment and Long term Evaluation of Survivorship; WCRF: World Cancer Research Fund; AASM&SRS: American Academy of Sleep Medicine and Sleep Research Society; PASE: Physical Activity Scale for the Elderly; DHD-15: Dutch Healthy Diet index-15; PSQI: Pittsburgh Sleep Quality Index; SCQ: Self-administered Comorbidity Questionnaire; PGI: Posttraumatic Growth Inventory; SCS-SF: Self-Compassion Scale—Short Form; MFI: Multidimensional Fatigue Inventory; HADS: Hospital Anxiety and Depression Scale; EORTC QLQ- C30: European Organisation for Research and Treatment of Cancer Quality Page 13 of 14 van Cappellen‑van Maldegem et al. 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https://openalex.org/W4300956466	https://zenodo.org/records/5958952/files/110-112.pdf	English	null	Studies on Arylacetamides. Part-II. Preparation and Antimicrobial Activity of p,p′-Bis(∞-carbarn oylarylm ethyl- hydrazinocarbonylmethoxy)diphenyl Sulphones	Zenodo (CERN European Organization for Nuclear Research)	1,989	cc-by	1,771	j, tndian CheBJ. Soe., Vol. 66, February 1989, pp. 110-112 j, tndian CheBJ. Soe., Vol. 66, February 1989, pp. 110-112 Studies on Arylacetami<iles. Part-11. Preparation and Antimicrobial Activity of p,p':.BIS( ct:-carbamoylarylmethyl- hydrazinocarbonylmethoxy )dipheny 1 Sui phones R. N. VANSDADIA, K. P. RODA and HANSA PAREKH* Department of Chemistry, Saurashtra University, Rajkot-860 000 Manuscripe received 7 March 1988, rwisetJ 30 October 1988, acce:~ted t1 November 1988 Some new arylacetamide derivatives hava been prepared bearing fl,fl'•dihydro:a:y- dipbenylsulphone moiety. fi,:P'-Bis(hydrazinocarbonylmetho:a:y)dfpbenyl salpbone was condensed with dift'erent aldehydecyanohydrin to get the respective nitriles. Tbe nitrites were then treated with concentrated sulphuric acid to get acetamide derivatives. The compounds were characterised by ir and pmr spectral data. Tbe products were also sc:reeaed for antimicrobial activity. (alongwith zone of inhibition in mm) R=phenyl (Sl. nos. 18- 26), cinnamyl (18- 23). 4-chlorophenyl (20-22), 2,6-dichlorophenyl (18-23), 4-methoxy- phenyl (19- 23), 4-nitrophenyl (18-28) and 2-hy- droxyphenyl (I 7- 23). In case of p,p -bis(<- carbamoylarylmethylhydrazinocarbonylmethoxy) di- phenyl sulphones (Table 1 ; Sl. nos. 24-46) ) maxi- mum activity was showed by those bearing R=2- chlorophenyl (SI. nos. 16-20), 2,4-dichlorophenyl (17-30), 2-methoxyphenyl (16-20). 4-methoxy- phenyl ( 18- 20), 3,4-dimethoxyphenyl ( 18- 23), 3- methoxy-4-hydroxyphenyl (17- 22), 4-nitrophenyl (16-20) and 2-hydroxy-3,5-dichlorophenyl (19- 20). A CETAMIDE derivatives have been found to possess wide biological activities. Several reports have appeared in the literature, which highlight their chemistry and uses1. The present communication deals with the preparation of some diarylacetamide of type (1) with an intention to synthesise better therapeutic agents. A C p,p'·Dihydroxydiphenyl sulphone condensed with ethyl chloroacetate, was treated with hydrazine hydrate to get p,p'-bis(hydrazinocarbonylmethoxy) diphenyl sulphone. The hydrazide was condensed with different aldehydecyanohydrin to get the nitrites. The nitriJes were then treated with con- centrated sulphuric acid to get arylacetamide deriva- tives. The structural assignments of the products were based on their elemental analysis, ir and nmr spectral data. The products were screened -for their antimicrobial activity. Experimental Melting points of the compounds were de mined in open capillary tubes and are uncorrected. Ir spectra (KBr) were recorded on a Shimadzu DR-1*" 435-IR spectrophotometer and pmr spectra on a XL-lOOA (100.1 MHz) spectrometer using TMS as internal reference. R-HC-HNHNOCH2CO~S02~0CHzCONHNH-CH-R teNH2 - - tONHz B-Aryl 1 p,p' -Bis(hydrazinocarbonylmethoxy )diphenyl sui- phone : An aqueous solution of hydrazine hydrate {7 ml, 80%) was added to a suspension of p,p'-bis (carbethoxymethoxy)diphenyl sulphone (8.45 g, 0.02 mol) in ethanol (15 ml). The mixture was reftuxed for 1 h and cooled to room temperature. The hydrazide thus separated on cooling was crysta- llised from DMF, (6.85 g, 87%), m,p. 228° (Found : C, 48.77; H, 4.57; N, 14.14. C18H 180aN•S requires: C, 48.72 i H, 4.60; N, 14.20%) ; vma:o: (KBr) 1 250 (C-0-C str., asym.), 1 145, 1 290 (S=O str., asym. and sym. respectively), 3 300 (NH or NHs str.) and 1 680 cm-1 {C=O str.) ; a (DMSO-d 8 ) 4.32 (4H, s, 2 x NH9 ), 4.6 (4H, s, 2 x HsCOAr), 8.0-6.96 (SH, m, 2 x ArH) and 9.4 (2H, s, 2xCONH). Antimicrobial activity : The antimicrobial screen- ing of the nitrile and amide derivatives was carried out using cup-plate method• at a concentration of 50 pg using gram-positive bacteria, i.e. Staphylococ- cus aureus, Staphylococcus citrus, gram-negative bacteria, i.e. Escherichia coli, Marcsane serratia and fungi Saccharomyces cerevisiae, Aspergillus niger. Activity of the compounds tested was determined by measuring the zone of inhibition. The results showed that most of the compounds are moderately active against difft!rent strains of bacteria and fungi (11- 20 mm, zone of inhibition). The most active among the p,p'-bis(<-cyanoaryl- metbylhydrazinocarbonylmethoxy)diphenyl sulpho- nes (Table I ; St. nos. 1- 23) were those bearing p,p'-Bis( <·cyanoarylmethylhydrazinocarbonylme- thoxy)diphenyl sulphone (Table 1 ; sl. no. I): To 110 VANSDADIA, RODA & PAREIKH STUDIES ON ARYLACBTAMIDBS. PART-II. TABI,JC 1-ANAI,Y~ICAI, AND PHYSICAl, DA~A 011 COMl'OUNDS BI. R M.p. Yield Mol. N%: no. ·o % formula Found/(Oalcd.) ~.p'-Bls{ aC.-cyanoarylmethylhydrazlnocarbonylmethoxy)diphenyl sui phones 1. Phenyl 158 '19 CaoH08N 80 0S 13.41 II. Olnnamyl 1103 611 OoeHuN.o.s (13 45) 111.44 3. 11-Furyl 172 159 CoeHuNoOwB (111.42) 13.'19 4. 2-Chlorophenyl 1106 77 c •• a •• m.N.o.s (18.90) 12.07 II, 4-0hlorophenyl 1137 80 c •• a •• OI.N.o.s (12.111) 111.10 6. 2,4-Dichlorophenyl 1153 84 c •• HuCI .. N.o.s (lUll) 11.00 7. 11,6-Dlohlorophenyl 218 74 CuH040l 4N.o.s (11.011) 11.06 8. 11-Methoxyphenyl 1154 66 c ... Experimental 1 310 (NH def.+CN str., amide-III sec.), 1 675 (C=O str., amide-I prim.), 1 640 (NH def. +CN str., amide-II prim.), 1 410 (NH def.+ CN str., amide-III prim.) and 3 400 cm-1 (NH or NHs str.) 1 tJ (DMSO-d 8 ) 3.86 (4H. s, 2x CONH 11), 4.78 (2H, s, 2xArCJl), 5.28 (4H,s,2xArOCH 11),6.94-6.02 {18H, m, 4x ArH), 8.50 (2H, s, 2x CHNH), 8.70 (2H, s, 2 x CONH) and 11.52 (2H, d, 2 X OH enolic). benzaldehyde (2.02 ml, 0.02 mol) dissolved in ethanol (20 ml), potassium cyanide(l3.02 g, 0.2 mol) dissolved in water (8 ml) was added followed by glacial acetic acid ( 12 ml}. The mixture was then stirred for five minutes to form aldehydecyano- hydrin and kept at 0°. p,p'-Bis(hydrazinocarbonyl- methoxy)diphenyl sulphone (3.94 g, 0.01 mol) in ethanol was added to the reaction mixture, contents kept for 24 h at room temperature and poured in to ice. The resulting solid was crystallised from ethanol. (4.93 g, 79%), m.p. 158° (Found : c. 61.47 ; H, 4.53; N, 13.41. CuHuN80 8S requires: C, 61.52; H, 4 51 ; N, 13.45%); !.>mas (KBr) 1 230 (C-0-C str., asym.), 1160, 1290 (S=O str., asym. and sym. respectively). 1 665 (C=O str., amide-I sec.), 1•575 (NH def.+CN str., amide-II sec.), 1 200 (NH def.+CN str., amide-III sec.). 2 260 (C=.N str., very weak band) and 3 400 cm-1 (amine sec.) ; 4(DMSO-d 6 ) 4.78 (2H. s, 2 x ArCH), 5.30 (4R, s, 2x Ar0CH 11), 5.92-8.02 (ISH. m,2x ArH),8.08 (2H, s, 2xCONHNH) and 11.52 (2H, d, 2xCONHNH, possibility of enolic OH also). Similarly, other nitrites were converted to the corresponding arylacetamides (Table 1 ; sl. nos. 24-46). Acknowledgement The authors are thankful to Prof. A. R. Parikh, Head, Department of Chemistry, Saurashtra University, for facilities and to the Gujarat State Government for awarding research scholarship to one of them (R.N.V.) and to U.G.C., New Delhi, for awarding Junior Research Fellowship to the other (K.P.R.). The authors are also thankful to the Director, I.I.T., Bombay for spectral data. Similarly, other aromatic aldehydes were con- densed with hydrazide (Table 1 ; sl. no. 1- 23). p, p' • Bis ( '-carbamoylarylmethylhydrazinocarbon- ylmethoxy )diphenyl sulphone (Table 1 ; sl. no. 24) : The p,p'· bis( c( -cyanoarylmethylhydrazinocarbony 1- methoxy)diphenyl sulphone (3.12 g, 0.005 mol) was dissolved in concentrated sulphuric acid {20 ml) at so and allowed to stand at room temperature for 48 h. The contents were poured into crushed ice and neutralised with ammonia. The product was isolated and crystallised from dioxane- D MF (3: 1), (2.28 g, 69%), m.p. 104° (Found : C, 58.15 ; H, 4.89 ; N, 12.68. CuHuN 8 0 11S requires: C, 58.18; H. 4.88; N, 12.72%) ; vm.., (KBr) 1 250 (C-0-C str .• asym.}, 1 140, 1 290 (S=O str., asym. Experimental a •• N.o .. s (11.011) 111.1111 9. 4-Methoxyphenyl IIlii 74 o ... H •• N.o.s (111.117) 111.20 10. 3,4-Dimethoxyphenyl 1147 '19 Oae HaoN 8 0 108 (12.117) 11.08 11. 3-Methoxy-4-hydroxyphenyl 234 69 CuHaoN 8 0 10S (11.28) 11.'15 111. ot-M ethoxy-3-hydroxyphenyl 1511 78 CuHaoNo010S (11. 72) 11.68 11J. 2-Nitropbenyl 1148 78 CuHuNa010S (11.72) 15.70 14. 3-Nitrophenyl 166 66 OuHorN110, 0S (15.68) 15.72 15. 4-Nitrophenyl 208 83 CuHuN80, 0S (15.68) 15.611 16. 11-Hydroxyphenyl 242 611 0 00H 29N 80 8S (15.68) 12.'17 17. 4-Hydroxyphenyl 182 70 0, 0H 29NoOaS (12.80) 12.'11 18. 2-Hydroxy-3,5-dichlorophenyl 181 74 CaoH 240l .. N 0 0 9 S (12.80) 10.62 2-Hydroxy-1-naphthyl 168 68 C40Ha.N 0 0 9 S (10.58) 19. 11.00 20. 2-Aminophenyi 196 60 Ca 0Ha 0 N 80 6S (11.10) 17:11! '.U. 3-Aminophenyl 186 58 CaoHo 0 N 80 8 S (17.11) 11.ma Ill. 4-Aminophenyl 188 65 Ca.HaoN 8 0 8 S (17.11) 17.09 4-Dimethy laminopbeny I 224 73 c •• H •• N.o.s (17.11) 28. 15.68 (15.'16) f'.'P'-Bis(ot-carbamoylarylmethylhydrazinooarbonylmethoxy)diphenyl aulpbones 114. Phenyl 104 69 CaaHaaNaOvS 111.68 115. Oinnamyl 156 59 o •• HaaN.o.,s (12.711} 1169 26. 2-Furanyl 182 54 OuHuN 00 10S (11.79) 13.0'1 27. II..Ohlorophenyl 1112 62 Oa~Ha 00l~N 8 0 8S {13.12) 11.56 4-0hlorophenyl 2118 70 CuHa 0 0l,N.,08 S (11.511) 118. 11.50 29. 2,4-Diohlorophenyl 207 72 c •• R 29Cl 4N 8 0 8S (11.52) 10.48 so. 2,6-Diohloropheny I 188 62 CuRuCI .. N.o.s (10.52) 10.46 31. 11-Metho:xyphenyi 251 61 OuHuN80 10S (10.52) 11.60 311. 4-Methoxyphenyl 2011 69 C-.R..,N80 10S (11 66) 11.62 88. 8,4-Dimethoxyphenyl 1100 '73 OaeH•oNeOuB (11.66) 10.78 34. S-Methoxy-4-hydroxyphenyl 198 59 a ... a .. N.o.,s (10.'16) 11.12 85. 4-:Methoxy-S-hydroxyphenyl 1155 68 o.,.u .. N.o ... s (11.16) 11.111 (11.16) 111 111 J, INDIAN CHBM. SOC., VOL. 66, FEBRUARY 1989 86. !a.Nitrophenyl 1!!2 8'1. 8-Nitrophenyl !a a sa. 4•Nltrophenyl 186 89. 2-Kydroxyphenyl 189 &0. t-Hydrox:yphenyl 198 41. 2-Hydrox:y-3.5-dlchlorophenyl 245 &2. 2-Hydroxy-1-naphthyl 205 48. 2-Aminophenyl 186 u. 3-Aminophenyl 190 45. 4-Aminophenyl 136 46. t-Dimetbylaminophenyl 25la (Table 1 contd.) 69 C88H 80N 80,.B 14.89 (14.92) 78 0 80H 80NaO,.S 14.87 CuBaoNaOuS (14.92) 77 14.96 69 0 12H 11NaO,oB (14.92) 12.08 Oa,HaoNaO,oB (lUS) '18 12.08 82 OaoHu014Na010B (BUS) 10.10 67 0 40HaeNaO,o8 (lO.lla) 10.62 (10.60) 62 OaoHuNsOsS 16.18 {16.22) 56 OuHuNaOaB 16.17 (16.22) 71 0 82HuNaOsS 16.28 (16.22) 79 0 88Hu'N80aB 14.98 {15.00) and sym. respectively), 1 675 (C=O str., amide-I sec.), 1 540 (NH def.+CN str., amide-II sec.). , , 2. F. KAVANAGH, "Analytloa.l Mioroblology", Academic, Ne'R York, 1963, p. 126. 1. W. GlCRHAR.D't and R. LJCHMANIIil, Ohem • .dbstr •• 198&, 101, 230159 ; A. K. EPSTICIN and B. &. HAIUUS, Ohem. .dbstr., 19£2, 36, 8904.; L. L'I.FON, Ohem. Abstr.,1984., 100, 2093'19; w. H. MII.I.AR, A. :M. Dassa't and R. Q. RIBX.IN, Ja., J. Am. Ohem. Soc., 1950, 72, '893 ; 8. L. DAI.AI. a.nd ;r, ;r, TatVlCDI, J, Ina1an Ohem. Soc., \960, 37, &87: P. DOMOJIT, J. Pha.rm. Balg., 1932, 14,157, 177, 198, 211, 249: L. ;r, KI&ltPA'tatca, Ohem • .dbstr •• 19'19, 90, 88928. References 1. W. GlCRHAR.D't and R. LJCHMANIIil, Ohem • .dbstr •• 198&, 101, 230159 ; A. K. EPSTICIN and B. &. HAIUUS, Ohem. .dbstr., 19£2, 36, 8904.; L. L'I.FON, Ohem. Abstr.,1984., 100, 2093'19; w. H. MII.I.AR, A. :M. Dassa't and R. Q. RIBX.IN, Ja., J. Am. Ohem. Soc., 1950, 72, '893 ; 8. L. DAI.AI. a.nd ;r, ;r, TatVlCDI, J, Ina1an Ohem. Soc., \960, 37, &87: P. DOMOJIT, J. Pha.rm. Balg., 1932, 14,157, 177, 198, 211, 249: L. ;r, KI&ltPA'tatca, Ohem • .dbstr •• 19'19, 90, 88928. 112
https://openalex.org/W4312734920	https://publicacoes.unifal-mg.edu.br/revistas/index.php/boletimalfenensedegeografia/article/download/1947/1477	Portuguese	null	A PARTICIPAÇÃO SOCIAL DAS MULHERES NO TERRITÓRIO DE IDENTIDADE SUDOESTE BAIANO	Boletim Alfenense de Geografia	2,022	cc-by	10,104	Resumo nense de Geografia v.2, n.4, 2022. Abstract A escolha da temática considera a necessidade de investigar a participação social da mulher dentro da perspectiva do desenvolvimento territorial no Território de Identidade Sudoeste Baiano – TSB. O trabalho tem como proposito contribui nas discussões a respeito da equidade de gênero e das novas formas de convívio e superação de situações historicamente construídas no espaço social da mulher. O artigo teve como aspectos metodológicos o acompanhamento das reuniões do colegiado territorial, das reuniões da câmara de mulheres, levantamento documental, entrevista semi-estruturada com a coordenadora da câmara de mulheres e registro fotográfico. A participação social de forma direta sobressai na construção de novos espaços que se dá a participação social e assim faz contribuições para esta promoção atividades significativas e tem contribuído na autonomia dos sujeitos sociais sobre a valorização do papel social das mulheres e participação social no território. Como resultados verificam que o TSB vivencia um processo de fortalecimento das mulheres na organização de empreendimentos, do envolvimento na formulação e acompanhamento das políticas públicas. The choice of theme considers the need to investigate the social participation of women within the perspective of territorial development in the Southwest Bahia Identity Territory - SBIT. The purpose of this work is to contribute to discussions about gender equity and new ways of living together and overcoming situations historically constructed in the social space of women. The article had as methodological aspects the follow-up of the meetings of the territorial collegiate, the meetings of the women's chamber, documental survey, semi-structured interview with the coordinator of the women's chamber and photographic record. Direct social participation stands out in the construction of new spaces that social participation takes place and thus makes contributions to this promotion of significant activities and has contributed to the autonomy of social subjects on the appreciation of the social role of women and social participation in the territory. As a result, they verify that the TSB is experiencing a process of strengthening women in the organization of enterprises, involving them in the formulation and monitoring of public policies. Key words: Territory, Territorial Development, Territory of Identity, Women, Social Participation A participação social das mulheres no território de identidade sudoeste baiano The social participation of women in the territory of southwest bahia identity La participación social de las mujeres en el territorio de identidad del suroeste baiano Boletim Alfenense de Geografia v.2, n Lucas Aguiar Tomaz Ferreira – lucasaguiar04@hotmail.com Mestrando em Geografia da Universidade Estadual do Sudoeste da Bahia – UESB. Orcid: https://orcid.org/0000-0002-0302-7079 Lucas Aguiar Tomaz Ferreira – lucasaguiar04@hotmail.com Mestrando em Geografia da Universidade Estadual do Sudoeste da Bahia – UESB. Orcid: https://orcid.org/0000-0002-0302-7079 Fernanda Viana de Alcantara – fernanda.alcantara@uesb.edu.br Professora Titular da Universidade Estadual do Sudoeste da Bahia – UESB. Orcid: http://orcid.org/0000-0002-9296-8558 m Alfenense de Geografia v.2, n.4, 2022. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Palavras-chave: Palavras-chave: Território, Desenvolvimento Territorial, Território de Identidade, Mulheres, Participação Social Desenvolvimento Territorial, Território de Identidade, Mulheres, Participação Social 53 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Introdução Ao observar o contexto histórico da sociedade brasileira, verifica-se que a mulher por longos anos não deteve papéis importantes, no entanto sua inserção no espaço social veio a acontecer por meio de movimentos sociais e diversas políticas públicas que se pode destacar, por exemplo, o direito ao voto assegurado em 24 de fevereiro de 1932 pelo decreto nº 21.076 instituído no código Eleitoral Brasileiro, consolidou-se na constituição de 1934 (voltadas exclusivamente para mulheres casadas, viúvas e solteiras) posteriormente estendido a todas pela Constituição Federal de 1946. Em 1983 nos estados de Minas Gerais e São Paulo surgiram os primeiros conselhos estaduais da condição feminina, no intuito projetar políticas públicas para as mulheres, sucessivamente em 1985 surge a primeira Delegacia de Atendimento Especializado à Mulher - DEAM. Boletim Alfenense de Geografia v.2, n Boletim Alfenense de Geografia v.2, n.4, 2022. Devido a esses e outros diversos avanços é importante destacar também as grandes conquistas adquiridas, mais recentemente, nos anos 2000, pode-se salientar a eleição da Senadora Marina Silva do Partido dos Trabalhadores - PT do Acre como um marco, onde a mulher ganha um papel destaque no âmbito político. Vale ressaltar também a criação da Lei nº 11.340 conhecida popularmente por Maria da Penha, promulgada em 7 de agosto de 2006, que tem por objetivo criar mecanismos para coibir a violência doméstica e familiar contra a mulher. Em 2010 tem-se outro grande marco nesse leque de progressos, trata- se do momento em que a primeira mulher ocupa a cadeira da Presidência do Brasil, Dilma Vana Rousseff. nense de Geografia v.2, n.4, 2022. Assim fica perceptível a necessidade de se discutir sobre o papel social da mulher, em especial no contexto do desenvolvimento territorial, e consequentemente nas discussões da ciência geográfica. Uma vez que compreender a inserção e a participação da mulher torna-se tema de interesse da geografia, visto que este debate pode ser compreendido como um fenômeno social, logo a geografia tem contribuições a trazer para a referida discussão. Desta forma, Santos expressa sobre o processo de (re)qualificação da ciência geográfica. A ciência geográfica está em constante processo de (re) construção, uma vez que, essa ciência não se preocupa apenas com a descrição de fatos ou fenômenos, mas com os processos que interferiu e ainda podem interferir na estrutura do espaço e da sociedade. Resumen La elección del tema considera la necesidad de investigar la participación social de las mujeres en la perspectiva del desarrollo territorial en el Territorio de Identidad del Suroeste de Bahía - TSB. El propósito de este trabajo es contribuir a las discusiones sobre la equidad de género y las nuevas formas de convivencia y superación de situaciones construidas históricamente en el espacio social de las mujeres. El artículo tuvo como aspectos metodológicos el seguimiento de las reuniones del colegiado territorial, las reuniones de la cámara de mujeres, levantamiento documental, entrevista semiestructurada con la coordinadora de la cámara de mujeres y registro fotográfico. La participación social directa se destaca en la construcción de nuevos espacios en los que la participación social se lleva a cabo y por ende hace aportes a esta promoción de actividades significativas y ha contribuido a la autonomía de los sujetos sociales sobre la valoración del rol social de la mujer y la participación social en el territorio . Como resultado, verifican que la TSB vive un proceso de fortalecimiento de las mujeres en la organización de las empresas, involucrándolas en la formulación y seguimiento de las políticas públicas. Palavras-chave: Territorio, Desarrollo Territorial, Territorio de Identidad, Mujeres, Participación Social Palavras-chave: Territorio, Desarrollo Territorial, Territorio de Identidad, Mujeres, Participación Social Recebido em: 20/07/2022 Aceito: 01/09/2022 Publicado: 03/10/2022 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 54 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Introdução Sendo assim, o seu conhecimento é fundamental, no que se refere à necessidade de conhecer o espaço geográfico, sua organização econômica, política, ideológica e social. Contribui de forma decisiva para a observação e 55 entendimento dos acontecimentos que se sucedem na sociedade. (V. SANTOS, 2015. p. 18/17) A respeito das preocupações da ciência geográfica com as questões sociais, torna-se possível refletir também a incontestável e notável função da mulher no território, desta maneira Horta et al julga que: “Adota-se a noção de território como uma construção social e, portanto, também política, além de um espaço em que a prática dos sujeitos sociais assume papel crucial na orientação do desenvolvimento.” (HORTA et al, 2014. p. 20). Boletim Alfenense de Geografia v.2, n Acerca da discussão do papel da mulher e o território, vale ressaltar que nos últimos anos, foi adotada no Brasil a política de desenvolvimento territorial, neste cerne a Bahia é um dos pioneiros neste processo, pois dividiu o estado, em 27 territórios, Tendo como critério para constituição as suas especificidades ou traços comuns, segundo a Superintendência de Estudos Econômicos e Sociais da Bahia- SEI, 2019. nense de Geografia v.2, n.4, 2022. Nesta direção, por meio das políticas públicas a mulher ganha maior autonomia, e passa a constituir uma nova territorialidade, além de fazer um resgate histórico no que diz respeito a desigualdades de gênero. Desta maneira, a mulher traz consigo uma dimensão simbólica de pertencimento com o lugar, diante disso são construídas as relações sociais por meio do espaço vivido onde são estruturadas as relações de poder, trabalho e outras. Todavia presente artigo é resultante de um processo de acompanhamento e observação do colegiado territorial do Território de Identidade do Sudoeste Baiano – TBS e da constituição de suas Câmaras Temática. Assim com exposto o recorte espacial para esta pesquisa é o TSB e como se dá o processo de participação das mulheres. O TSB é composto por 24 municípios e tem município de Vitória da Conquista como sede, para melhor articulação do Colegiado do Território Sudoeste Baiano, o referido território foi dividido em três polos, Condeúba, Poções e Vitória da Conquista, o que facilitou as atividades dentro do mesmo. Para uma melhor compreensão da localização do território, segue o mapa da figura 1. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Introdução 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 56 g FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 56 56 Figura 1 - Território de Identidade Sudoeste Baiano, 2019. Elaboração: Ferreira, 2019. Boletim Alfenense de Geografia v.2, n Boletim Alfenense de Geografia v.2, n.4, 2022. m Alfenense de Geografia v.2, n.4, 2022. enense de Geografia v.2, n.4, 2022. Elaboração: Ferreira, 2019. O TSB conforme se observa na figura 1 apresenta 24 municípios e apresenta a área de 26.809,99 km² com a população de 698.719 habitantes sendo 345.444 a população masculina e 353.275 a população feminina. A demografia do território é aproximadamente de 25,9 hab/km², segundo a SEI, 2019. Assim como exposto anteriormente este texto foi fruto do processo de acompanhamento e observações das plenárias do colegiado territorial do TSB, bem como, a constituição e articulação de suas Câmaras Temática e com as colocações, intervenções e leituras da disciplina Território Cultura e Ruralidades, Ministrada pelo Prof. Dr. Flamarion Dutra Alves do Programa de Pós-Graduação em Geografia da Universidade Federal de Alfenas – UNIFAL que possibilitou fazer um o resgate da do debate questão de gênero dentro da articulação do espaço de participação TSB. No desenvolvimento metodológico do presente artigo realizou-se levantamento bibliográfico a respeito da temática. Participou-se dos eventos direcionados ao fortalecimento da participação social da mulher no território, tais como: das reuniões da Câmera Temática de Mulheres do Território, do I Seminário de Mulheres em 2017 e acompanhamento das plenárias do Colegiado Territorial, entrevista com a coordenadora da Câmara de Mulheres do TSB. 57 Desenvolveu-se levantamento documental, onde foram analisadas as listas de frequências das reuniões do colegiado e demais espaços de participação e ainda tabulados os dados ao observar o número de representantes mulheres em diferentes momentos e reuniões com registros fotográficos. Desenvolveu-se levantamento documental, onde foram analisadas as listas de frequências das reuniões do colegiado e demais espaços de participação e ainda tabulados os dados ao observar o número de representantes mulheres em diferentes momentos e reuniões com registros fotográficos. [...] O território se apóia no espaço, mas não é o espaço. É uma produção a partir do espaço. Ora a produção, por causa das relações que envolvem que se inscreve num campo de poder. Produzir uma relação do espaço já uma apropriação [...], portanto, mesmo se permanece nos limites de um conhecimento. Qualquer projeto de no espaço é expresso por uma representação revela a imagem desejada de um território, de um local de relações. (RAFFESTIN, 1980, p.144) O espaço, território e a discussão de gênero O espaço geográfico está em constante transformação tanto em seus aspectos físicos quanto sociais. É dever da Ciência Geográfica analisar e compreender a dinâmica dos processos que envolvem a produção e a (re)produção do espaço geográfico. Neste sentido, não se tem um espaço congelado, e sim dialético e produzido e (re)produzido por meio das relações da sociedade e natureza com expressa Moreira (1994). E por meio deste processo dialético entre o espaço e tempo se obtém transformações socioespaciais em diferentes estágios e em várias formas sobre a relação intrínseca da sociedade e natureza essas várias relações que produz e (re)produz o espaço geográfico se perpetuam como afirma SANTOS (2006). enense de Geografia v.2, n.4, 2022. Dentre as categorias de análise da geografia, o território oferece a capaz de sustentar as discussões teóricas para arrazoar a participação social das mulheres no TSB. Nesta direção, Raffestin (1980 p.143) propõe a dissociação do espaço e território, “Espaço e território não são termos equivalentes” e define “É essencial compreender bem que o espaço é anterior ao território. O território se forma a partir do espaço, é resultado de uma ação conduzida por um ator sintagmático [...] em qualquer nível.” (RAFFESTIN 1980 p.143). O autor contribui ao afirmar que o espaço tem relação com tudo que é produzido e o território está ligado com a relação de poder. Como se pode observar na citação de Raffestin: FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 8 Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Gimenez (2000) vê o território como o resultado de uma apropriação e valorização do espaço de imediato representado e promovida por meio do trabalho e prevê assim como Souza (1995) um campo de forças que perpassa do físico ao ideológico, ou até mesmo sem materializado simbólico, ou melhor, imaterial: 59 [...] el territorio sería el resultado de la apropiacion y valorización de espacio mediante la representación y el trabajo, una produción a partir del espacio inscripto en el campo del poder por las relaciones que pone en juego; y en cuanto se caracterizaria por su “valor de cambio” y podria representarse metafóricamente como “la prision que nos hemos fabricado para nosotros mismos”.( GIMENEZ, 2000, p. 22) Tanto para Gimenez quanto para Souza apontam as “facetas” do território, pois pode ser representado por dimensões culturais, sociais, econômicas e naturais essa concepção de território corrobora com o pensamento de Haesbaert (2004). Ainda sobre a culminância de pensamentos dos autores eles têm a concepção de que os territórios podem atuar em diferentes escalas, e neste sentido Gimenez (2000, p. 24) expressa sobre a pluralidade de atuação do território. “Asi definido, el territorio se pluraliza según escalas y niveles historicamente constituídos y sedientos que van desde lo local hasta lo suparnacional, pasando por escalas intermedias como las del municípios o comina, la region, la província, y la nacion.” Boletim Alfenense de Geografia v.2, n.4, 2022. enense de Geografia v.2, n.4, 2022. Por sua vez, Rocha (2010) aborda o território como uma categoria indispensável para vida cotidiana humana, pois é entendida como existência do poder em várias escalas, desde local até mais abrangente, a global, podendo fazer a relação Local-Global-Local. Ainda no intuito de conceituar a categoria território em várias contribuições vale destacar a contribuição de Horta et al (2014, p. 20) que salientam que: “Adota-se a noção de território como uma construção social e, portanto, também política, além de um espaço em que a prática dos sujeitos sociais assume papel crucial na orientação do desenvolvimento.”. Ornat e Silva (2014) têm uma concepção de território está ligado ao poder e a soberania das relações sociais materiais e imateriais e não é as relações de poder que vão configurar por si só o território, mas as reflexões desta relação se configuram-se um território. Atualmente a utilização do termo território não pertence apenas a Geografia. Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Também neste cerne de definir o território por meio das relações sociais, cipalmente a importância dele para essas atividades. “[...] o território é fonte 58 de recursos e só assim pode ser compreendido quando enfocado em sua relação com a sociedade e suas relações de produção, [...], ou seja, pelas diferentes maneiras que a sociedade se utiliza para se apropriar e transformar a natureza” (SPOSITO, 2004, p. 112-113). Para o geografo Corrêa (1996. p. 62) aborda o caráter político da categoria desta categoria de analise: “O território é a ação social (expressão política) implica a organização dos componentes do espaço geográfico em processos concretos que procuram alcançar os objetivos comuns.” É notório que o território não está desvinculado do espaço e sim é uma parte dele, onde se pode observar de melhor maneira a interação da sociedade com a natureza, transformando, ou melhor, (re)configurando esses territórios para sua existência e por virtude disso gera os conflitos. enense de Geografia v.2, n.4, 2022. Haesbaert (2004) considera que o território apresenta três vertentes basilares, a primeira de cunho político, quando está diretamente ligada à relação de poder, neste sentido seria uma concepção mais delimitada do conceito território na qual a categoria é vista como um espaço controlado e delimitado, no que emana é o poder, que na maioria das vezes é relacionado ao poder político estatal. Na segunda, o autor aborda uma vertente culturalista ou simblólico- cultural, em que o espaço é visto através da priorização da dimensão simbólica tornando em algo subjetivo. É conceituado como um fruto da apropriação ou valorização de um grupo em relação ao espaço vivido. A terceira e última vertente considera-se mais economicista, pois, nessa concepção é enfatizado as relações econômicas, por esta perspectiva, é entendido como uma fonte de recurso, para melhor compreensão desta vertente, a exemplo do debate da luta de classes sociais e a relação capital-trabalho, resultando na divisão territorial do trabalho. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 20 ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Hoje a categoria território é utilizada como unidade de planejamento pelo estado, isso não é exclusividade apenas do Brasil, outros países da América Latina e Europeus utilizam a referida categoria. Em especial, o Brasil a partir da década de 1990 adota o território como unidade de planejamento para execução de políticas públicas. 60 A respeito desse uso do território para o planejamento Perico (2009. p.26) define - o “[...] como a dimensão política do espaço – reconhecido e identificado – como uma unidade da gestão política que distinguir e a ele atribui existência, de certa forma, institucionalizada. Nem sempre o território se constitui em uma entidade territorial, como município, província, departamento ou estado.”. O autor aponta a maneira que o Estado concebe o território que é delimitado por algo em comum, seja relações econômicas, culturais e dentre outras, então ele diz que: “É suficiente ser reconhecido como unidade que pode controlar ou interagir [...]. Logo pode ser a bacia de um rio, a união de organizações [...], um espaço com nítidas características étnicas ou um espaço definido por redes econômicas bem caracterizadas. (PERICO, 2009, p. 26) Boletim Alfenense de Geografia v.2, Esse contexto revela novos desafios para as discussões a respeito do território tanto para a Ciência Geográfica, bem como para outras áreas do conhecimento e órgãos de planejamento que passam a utilizar a categoria. A utilização do território como unidade de planejamento, traz à tona novas contribuições à concepção de território, nesta quadra, Dias (2016) apresenta a categoria, conforme a abordagem da Secretaria de Desenvolvimento Territorial - SDT (2003): nense de Geografia v.2, n.4, 2022. [...] espaço físico, geograficamente definido, não necessariamente contínuo, caracterizado por critérios multidimensionais, tais como o ambiente, a economia, a sociedade, a cultural, a política e as instituições, e uma população, com grupos sociais relativamente distintos, que se relacionam internamente e externamente por meio de processos específicos, onde se pode distinguir um ou mais elementos que indicam identidade e coesão social, cultural e territorial. (DIAS, 2016, p. 27) No contexto político-institucional da SDT (2003), Perico (2009) e Dias (2016) acrescentam que a identidade se expressa como uma forma de territorialidade que demonstra o sentido de formação política de cada indivíduo e que a vontade coletiva somados tem o resultado de nacionalistas, patrióticos, regionalistas, um amor pelo espaço vivido e várias manifestações da força social. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 [...] espaço físico, geograficamente definido, não necessariamente contínuo, caracterizado por critérios multidimensionais, tais como o ambiente, a economia, a sociedade, a cultural, a política e as instituições, e uma população, com grupos sociais relativamente distintos, que se relacionam internamente e externamente por meio de processos específicos, onde se pode distinguir um ou mais elementos que indicam identidade e coesão social, cultural e territorial. (DIAS, 2016, p. 27) Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Ainda a respeito da utilização da categoria para o planejamento e como unidade de execução das políticas públicas, Alcantara (2013) afirma: Pontua-se que as categorias de estudo da geografia vêm sendo utilizadas e direcionadas para o ato de planejar [...] e merece destaque o território. Atos diretamente vinculados à [...] reprodução do homem no espaço, e logo construídos temas de investigação recai sobre a perspectiva da análise geográfica. (ALCANTARA 2013 p. 84/85) 61 A categoria é escolhida para essa nova forma de planejamento, pois dentro da perspectiva da gestão das políticas públicas, o território abraça a relação sociedade e natureza de forma diferenciada das demais categorias já que nela contém nitidamente a relação de poder, cultural e econômica. Para Haesbaert (2010) a utilização da categoria território para implementação de políticas públicas não se restringiu apenas ao Estado brasileiro, outros países o adotaram como forma de ordenamento e planejamento tais como: Portugal, Espanha, França e dentre outros. Vale ressaltar que Portugal tem seu próprio ministério para tratar de assunto relacionado ao ordenamento e ao desenvolvimento regional, denominado Ministério do Ambiente. Desta forma, conforme afirmação de Haesbaert (2010, p. 157), “reunindo ao mesmo tempo o “ambiente”, o território e a região, três conceitos geográficos fundamentais”. Boletim Alfenense de Geografia v.2, n Boletim Alfenense de Geografia v.2, n.4, 2022. A emersão da discussão sobre o território apresenta a questão do espaço vivido e a relação de pertencimento, pode-se caracterizar a identidade. Neste sentido, Alcantara (2013, p 95) enfoca que “o debate acerca da abordagem territorial sugere a retomada de outros debates, como o do planejamento e em particular o desenvolvimento”. Para melhor compreensão sobre a abordagem territorial vale salientar a contribuição de Saquet: nense de Geografia v.2, n.4, 2022. A abordagem territorial é uma forma de compreensão [...] pode contribuir na superação de aspectos das dicotomias natureza/sociedade e idéia/matéria e subsidiar a elaboração de proposta de desenvolvimento, valorizando a relações sociais entre os sujeitos. (SAQUET, 2010, p.177) Sobre a contribuição da análise do desenvolvimento sobre a concepção de Alcantara (2013) através do advento das crises econômicas e das grandes reformas neoliberais ocorridas no Brasil de 1990, nas últimas décadas o país recebe uma atenção focalizada, dando precedentes para novas experiências destacando-se a valorização da diversidade, tais como: 1- Econômicas; 2 – Sociais e 3- Políticas. Pode-se ser definido como expressa Alcantara (2013 p.95) “multidimensionalidades dentro do âmbito do desenvolvimento.”. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Vale destacar que é significativo o período em que Brasil vem trabalhando com a categoria território dentro da perspectiva territorial, de acordo com Haesbaert: Em termo de políticas oficiais o termo acabou sendo central em propostas como PNOT - Política Nacional de Ordenamento Territorial, vinculada ao Ministério de Integração Nacional (MIN), vinculada a partir de 2004, o PTC - Programa de território da Cidadania do Ministério Agrário (MDA) criado em 2008 e 62 recentemente o PEEITE Programa de Educação Escolar Indígena e seus Territórios Educacionais, vinculado ao Ministério da Educação do Ministério da Educação (MEC). (HAESBAERT 2010, p.156) recentemente o PEEITE Programa de Educação Escolar Indígena e seus Territórios Educacionais, vinculado ao Ministério da Educação do Ministério da Educação (MEC). (HAESBAERT 2010, p.156) O estudo do território na perspectiva da política de desenvolvimento territorial vem ganhando a atenção dos estudiosos das diferentes áreas do conhecimento e também das esferas estatais, destaca-se o poder municipal, pois o planejamento ficou mais abrangente, ao atender o maior número de pessoas possíveis, especialmente por tratar das questões relacionadas à criação dos chamados espaços de participação. É importante ressaltar que essa ideia de desenvolvimento vai muito além daquele desenvolvimento econômico e sim um desenvolvimento social, como aborda Sen (2010). Por isso, vale destacar a criação dos colegiados territoriais, e de forma mais específica no estado da Bahia, em seu estudo a respeito do colegiado do Território de Identidade Sudoeste Baiano, V. SANTOS (2015) afirma que: Boletim Alfenense de Geografia v.2, n nense de Geografia v.2, n.4, 2022. O Brasil é um país onde a desigualdade social é um fato materializado em todo território. Sendo assim, há necessidade de criar estratégias políticas que visem à superação das demandas, impasses e problemas no país. Nesse sentido, a abordagem territorial ganha relevância, as políticas de desenvolvimento territorial emergem com ênfase nas relações de pertencimento e identidade que a população tem com um determinado espaço. Ao longo da história brasileira ocorreram inúmeras tentativas no propósito de promover uma melhor distribuição da renda e de reduzir as desigualdades sociais e econômicas. Mas, na maioria das vezes as políticas são frágeis no seu processo de formulação e concretização. As políticas públicas que são desenvolvidas em âmbito nacional nem sempre se adequam as particularidades existentes no país. (V. , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2 ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte SANTOS, 2015, p.30) E por sua vez, o planejamento precisa ser discutido dentro dos denominados espaços de participação social, tais como: Conselhos Municipais, Colegiados Territoriais, Câmaras Temáticas e Técnicas dentre outros. Nesse sentido, Dias (2016. p 27) diz que “O objetivo primordial da SDT [...] é dar sustentação a um processo em que a preocupação social se entregaria às questões econômicas, onde a distribuição da renda e ganhos sociais pudesse adquirir uma dinâmica comum [...]”, assim o autor prever que o desenvolvimento territorial com foco no local se concretizava por meio da mobilização e articulação dos aspectos econômicos, sociais, ambientais e políticos. Neste sentido, os espaços de participação social previsto pela SDT recaem no conceito de “espaço público” de Arendt (2010), que e idealizado um espaço de 63 participação política que visa à pluralidade e a liberdade de um determinado sujeito. O primeiro conceito está ligado à condição de cada sujeito como ser único, e deve-se respeitá-lo em sua condição antagônica, assim, cada ser teria direito de fala, se impor e pedir direito de na participação Política. Já o conceito de liberdade significa a manifestação voltada para o processo democrático de um espaço público e pode ser definida como liberdade de opinião, crença, manifestação, e equidade no processo de discussões das liberações de ações democráticas. Boletim Alfenense de Geografia v.2, n No que diz respeito à abordagem promulgada pela SDT, o estado da Bahia foi um dos primeiros a articular a abordagem do desenvolvimento territorial, com a constituição dos seus Territórios de Identidade. É importante frisar que essa territorialização culmina com as políticas públicas do Governo Federal tais como os territórios Rurais e os territórios da Cidadania. A Bahia sendo um dos primeiros neste processo como tentativa de alcançar o seu desenvolvimento, a Superintendência de Estudos Econômicos e Sociais da Bahia – SEI dividiu a Bahia em 27 territórios de identidade. enense de Geografia v.2, n.4, 2022. O Colegiado Territorial, neste cenário, é o principal mecanismo de participação dos sujeitos sociais do território, desta forma, é denominado espaço de conflitos de interesses. Segundo Alcantara (2013) o colegiado é “Campo de Forças” e Leite (2009) denomina como “arenas”, assim, reforça o conceito da natureza do território que é ligado ao de poder. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Neste sentido, se faz relevante estudar de que maneira são conduzidas as discussões e debates sobre a questão de gênero nestes espaços, uma vez que de acordo Beauvoir: O mundo sempre pertenceu aos machos. Nenhuma das razões nos propuseram para explicá-lo nos pareceu suficiente. É revelada à luz da filosofia existencial os dados da pré-história e da etnologia que poderemos compreender hierarquia do sexo se estabeleceu. (BEAUVOIR,1939, p.81) E com a ciência Geografia não poderia ser diferente como expressa Silva: A razão de suas ausências dentro do discurso geográfico deve ser entendida pela legitimação naturalizada dos discursos hegemônicos da geografia branca, masculina e heterossexual, que nega essas existências e também impede o questionamento da diversidade dos saberes que compõem as sociedades e suas variadas espacialidades. (SILVA, 2009, p. 26) FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 64 Ao analisar o pensamento de Silva, constata-se que a discussão do saber geográfico se constituiu durante muito tempo por meio da hegemonia da masculinidade e da heterocisnormatividade que se tem relação intrínseca ao poder. Neste sentido, a autora expressa a existência e a atividade de luta para incluir o debate de gênero ao debate e analise da Geografia, sobretudo, a Geografia Brasileira: [...] Não podemos deixar de questionar a impermeabilidade da geografia brasileira a esse movimento político-científico e, acima de tudo aguçar nosso senso crítico para compreender que os princípios que organizam a produção discursiva da ciência hegemônica são os mesmo que produzem os silêncios e as ausências. (SILVA, 2009, p. 27) [...] Não podemos deixar de questionar a impermeabilidade da geografia brasileira a esse movimento político-científico e, acima de tudo aguçar nosso senso crítico para compreender que os princípios que organizam a produção discursiva da ciência hegemônica são os mesmo que produzem os silêncios e as ausências. (SILVA, 2009, p. 27) Boletim Alfenense de Geografia v.2, n.4, 2022. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no Também neste cerne de definir o território por meio das relações sociais, principalmente a importância dele para essas atividades. “[...] o território é fonte Boletim Alfenense de Geografia v.2, n É importante frisar que a discussão de gênero dentro da Geografia se dá em virtude da corrente de pensamento Humanista ou Cultural que reconheceu como parte das análises desta ciência o movimento feminista, neste sentido, afirma a diferença existente no espaço ligado as relações entre corpos, identidade e poder na produção do espaço (i) material (SILVA 2009). Nos dias atuais, aos poucos, mulheres vem ocupando espaços cada vez mais. Esses grupos de mulheres organizadas estão buscando direitos e idealizando políticas públicas que podem contribuir para amenizar as disparidades existentes no contexto social brasileiro. Desse modo, reforça-se a contribuição de Silva (2009) sobre o quão é importante debater gênero na ciência geográfica, pois, a luta para reconhecimento de respeito é um fenômeno existente na produção do espaço geográfico, o objeto de estudo da ciência. E analisar a produção do espaço por meios desses sujeitos sociais para entender a condição espacial expressa por Carlos (2011) e que espaço é meio, condição e produto das relações sociais. Neste sentido, é necessário se pensar em uma “metageografia” como expõe Carlos (2011) que faça um reconhecimento de fenômenos que acontece mo espaço geográfico para sua produção e (re)produção. A configuração que se dá sobre o território na visão do planejamento abordado pelo Estado é o mesmo defendido por Raffestin (1983), pois nesta discussão de implementação de políticas públicas acaba sendo beneficiado o segmento que tem mais força, neste sentido, reforça a disparidade na discussão de gênero. Destarte, a abordagem territorial utilizada pelo Estado não compreende a totalidade desta categoria para a Geografia e a utiliza de maneira 65 deturpada como sinônimo de regionalização corroborando com a corrente tradicional da ciência geográfica. No entanto, é importante para os estudos geográficos, a análise crítica das “novas” performances sociais com base nas políticas públicas inseridas no contexto territorial. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Processo de formação e funcionamento da câmara de gênero e a participação social no TSB Boletim Alfenense de Geografia v.2, n Para melhor funcionamento do Colegiado Territorial do Sudoeste Baiano foram criados espaços discussões, articulação e planejamento das políticas públicas no TSB. São quatro câmaras temáticas: 1) Inclusão Produtiva - que trata de assuntos ligados à produção, sobretudo, a produção agrícola. -; 2)Cultura - que vai abarcar as questões culturais existentes, pois, o TSB é um celeiro de artistas. -; 3) Juventude - o Território um índice de população de jovem expressiva e eles carecem de políticas públicas que possam atender suas necessidades tanto no rural quanto no urbano. - e 3) Mulheres - o espaço relacionado a discutir gênero no território, visto que o TSB possui mais de 50% da sua população é feminina isso dados do último censo do IBGE (2010) e último levantamento feito pela SEI (2016) para traçar os perfis do Território de Identidade da Bahia. Boletim Alfenense de Geografia v.2, n.4, 2022. enense de Geografia v.2, n.4, 2022. As Câmaras Temáticas do TSB foram constituídas nos meados de setembro de 2015 em uma reunião ordinária da plenária, cuja reunião pode contar com representantes da SDT, SPM e demais Secretarias do governo estadual. Não podendo esquecer a presença do NEDET que foi de grande ajuda para o colegiado territorial. Para entender todo o processo de construção e funcionamento da Câmara de Gênero entrevistou-se a atual coordenadora. Ela assumiu a coordenação há pouco tempo, porém faz parte da câmara desde sua fundação. Criada em 9 de setembro de 2015, com 9 participantes, conforme apresenta a figura 2. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 66 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 66 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 66 Figura 2: Lista de membros no momento da criação da Câmara de Mulheres do Território Sudoeste Baiano, 2015. Figura 2: Lista de membros no momento da criação da Câmara de Mulheres do Território Sudoeste Baiano, 2015. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no erritório de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764 1422 DOI: https://doi org/10 29327/243949 2 4 4 g ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Processo de formação e funcionamento da câmara de gênero e a participação social no TSB Figura 2: Lista de membros no momento da criação da Câmara de Mulheres do Território Sudoeste Baiano, 2015. Fonte: Acervo do NEDET, 2015 Fonte: Acervo do NEDET, 2015 Boletim Alfenense de Geografia v.2, n Boletim Alfenense de Geografia v.2, n.4, 2022. enense de Geografia v.2, n.4, 2022. Fonte: Acervo do NEDET, 2015 A Câmara temática se trata de um grupo que tenta atender à diversidade do território, dos seus 24 municípios, para desenvolver de suas ações e ainda se planeja em desenvolvimento de forma abrangente, pois ele se constitui por pessoas de dois segmentos, do poder público e sociedade civil, apesar de que em sua constituição está bem mais frequentes membros dos Municípios de Vitória da Conquista e Barra do Choça. Este grupo é mais uma estratégia do Estado para promover a participação social dos sujeitos sociais e acarretado a isso se discutir e implementar as políticas públicas. Com base nas pesquisas documentais ainda não se tem um regimento interno do grupo. Este espaço de participação social é um espaço democrático que dá o direito de fala a todos os componentes. A câmara, hoje, em 2020, possui 30 mulheres de quase todos os municípios que compõe o TSB, é um número relativamente representativo, pois se sabe que poucas pessoas conseguem dedicar seu tempo às atividades que não darão um recurso financeiro, porém se 67 envolver nesta perspectiva do desenvolvimento territorial o indivíduo recebe mais que recursos financeiros, recebe qualidade de tentar diminuir as mazelas sociais existentes no país. Para fazer parte da Câmara temática de mulheres é algo fácil, segundo a coordenadora, basta residir em um dos municípios que compõe o Território de Identidade Sudoeste Baiano e se interessar nas discussões que são feitas na câmara. Algo importante é que a câmara está aberta ao diálogo em discutir outras temáticas para além das mulheres cisgênero, a exemplo, as questões de Gênero e Sexualidades como os LGBTQIA+. Boletim Alfenense de Geografia v.2, n Um fator relevante é a promoção de eventos realizados no TSB, seja de ação o próprio Colegiado ou de ações do Núcleo de Pesquisa e Extensão em Desenvolvimento Territorial – NEDET da Universidade Estadual do Sudoeste da Bahia – UESB, o que demonstra uma inquietação para uma busca do desenvolvimento. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Processo de formação e funcionamento da câmara de gênero e a participação social no TSB Pode-se observar que há uma participação expressiva deste evento e vale destacar aqui o I Encontro Territorial de Mulheres realizado pelo Núcleo da UESB, conforme Figura 3, e o primeiro encontro de Mulheres no Polo de Condeúba realizado no Município de Cordeiros, na figura 4: Figura 3 - I Encontro territorial de Mulheres do Território de Identidade do Sudoeste Baiano- TSB na Universidade Estadual do Sudoeste da Bahia - UESB em 2017 Fonte: Acervo do NEDET, 2017. Figura 3 - I Encontro territorial de Mulheres do Território de Identidade do Sudoeste Baiano- TSB na Universidade Estadual do Sudoeste da Bahia - UESB em 2017 Fonte: Acervo do NEDET, 2017. 68 Figura 4 - I Encontro e de Mulheres do Polo de Condeúba realizado no Município de Cordeiros/BA em 2018 ra 4 - I Encontro e de Mulheres do Polo de Condeúba realizado no Município de Cordeiros/BA em 2018 Fonte: Acervo do NEDET, 2018. Boletim Alfenense de Geografia v.2, n enense de Geografia v.2, n.4, 2022. Fonte: Acervo do NEDET, 2018. Também pode-se observar a importância destes eventos para empoderamento destas mulheres, pois é por meio destes espaços que se abre um leque de oportunidades para as pessoas, mostrando uma realidade que até então muitas delas desconhecem. Outro fator relevante é que a Câmara não possui uma sede própria; é uma organização que tem a necessidade de circular por todo território para se obter uma visão mais ampla do território e ela se reúne mensalmente em locais que os membros tentam eleger durante a reunião anterior, isso faz com que quebre um com a lógica da centralização de poder nos municípios com mais desenvolvimento econômico, no caso do TSB, o Município de Vitória da Conquista. Processo de formação e funcionamento da câmara de gênero e a participação social no TSB Outro fator relevante é as componentes dos comitês foram direcionadas e assessoradas pelo NEDET com o apoio do Colegiado - CODETER, para estarem presentes nas audiências de cunho decisivo referentes aos orçamentos destinados a políticas públicas para mulheres, com esses incentivos pode-se destacar: a realização de dez Conferências Municipais de Políticas Públicas para Mulheres no Território; Criação 10 Comitês Municipais de Mulheres; a promoção de discussões nos municípios para criação dos Conselhos de Direito das Mulheres 69 nos municípios do Território; Levantamento e atualização dos dados do colegiado, de modo especial, os dados referentes à participação e atuação das mulheres nesse espaço; Formação de um banco de dados das secretarias de assistência social e lideranças que trabalham com mulheres nos municípios. E mapeamento de habilidades destas mulheres para criar formas de capitalização de recursos para que elas tenham de fato uma autonomia financeira. Na perspectiva de fortalecer a participação da mulher no colegiado territorial, verificou-se a existência de um número considerável de mulheres, participantes das plenárias e discussões, em torno 30 mulheres. Segundo a análise, foi possível notar que 50% dos membros participantes das reuniões do colegiado são mulheres. Ainda dentro desta perspectiva pode-se salientar que o colegiado é coordenado por uma figura feminina. Levando em conta que o colegiado é um composto de 100 organizações, dentre essas pode se perceber que 16 tem representatividade da mulher e estas merecem destaque: os sindicatos de trabalhadores e trabalhadoras rurais de alguns municípios, tais como: Poções, Anagé, Condeuba, Belo Campo, Tremedal e dentre outros; e a União Brasileira de Mulheres - UBM, destas 16% compõem o CTSB de mulheres, vale ressaltar que essas instituições são da Sociedade Civil, e ainda não se tem nenhuma do Poder Público. Boletim Alfenense de Geografia v.2 enense de Geografia v.2, n.4, 2022. Ao considerar a importância da participação feminina em todos os setores da atividade socioeconômica, ampliando assim a possibilidade de emprego e renda para as mulheres, jovens e adultos do TSB, e a formação de lideranças nos municipais, o trabalho tem buscado organizar e estudar essa participação no TSB. Desta maneira, é de grande relevância a realização de diversas ações, no sentindo de promover intervenções nos vinte quatro municípios do território para manter e fortalecer o processo de empoderamento da mulher no território. , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Processo de formação e funcionamento da câmara de gênero e a participação social no TSB Nos anos de 2019 houve eventos promovidos pela Câmara de Mulheres do TSB sobre o papel da mulher no contexto da Agricultura familiar agregando mulheres dos 24 municípios. Houve a contribuição da Câmara no II Seminário Perspectivas da Juventude em 2019 (Observar figura 5) que visa traçar planos estratégicos e ações para a juventude, a câmara de mulheres estava presente articulando o debate de gênero e sexualidade para dá visibilidade a estes públicos tão esquecidos na sociedade brasileira. Figura 4 – II Seminário Perspectivas Para Juventude no momento do Diálogo sobre Gênero e Sexualidade realizado na Universidade Estadual do Sudoeste da Bahia – UESB em novembro de 2019. Fonte: Acervo do NEDET, 2018. Figura 4 – II Seminário Perspectivas Para Juventude no momento do Diálogo sobre Gênero e Sexualidade realizado na Universidade Estadual do Sudoeste da Bahia – UESB em novembro de 2019. Figura 4 – II Seminário Perspectivas Para Juventude no momento do Diálogo sobre Gênero e Sexualidade realizado na Universidade Estadual do Sudoeste da Bahia – UESB em novembro de 2019. Boletim Alfenense de Geografia v.2 Boletim Alfenense de Geografia v.2, n.4, 2022. enense de Geografia v.2, n.4, 2022. Fonte: Acervo do NEDET, 2018. Nos dias atuais toda a atividade dentro de território está sendo desenvolvida de forma remota em virtude do cenário da pandemia do Covid-19, isso dificulta o processo de articulação, pois por se tratar de um território com características rurais e por tanto com dificuldade de acesso internet e outros meios de reunião no contexto da pandemia. Em 2020 a Câmara temática de mulheres se encontra desarticula, pois não está conseguindo desenvolver ações de forma remota, pois não está conseguindo as ações de forma remota. Apesar dos desafios, registram-se avanços principalmente sobre o empoderamento da mulher e que ela possa assumir outros papéis sociais, outros espaços, às vezes mulheres que antes não tinham acesso. As políticas públicas de gênero ainda necessitam muito da colaboração e união das mulheres e é importante de incluir as minorias na discussão do desenvolvimento territorial e a existência de entidades específicas no poder público do TSB. A partir dessa preocupação para incorporar o debate sobre os gêneros e principalmente, sexualidade dentro das discussões do TSB, visto que esses 71 temários sempre estiveram subalternizados, as margens da sociedade brasileira, isso não é exclusividade do deste território, mas de outros do Brasil. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no erritório de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 , g ; , p p ç identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764 1422 DOI: https://doi org/10 29327/243949 2 4 4 tidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. ISSN: 2764-1422. DOI: https://doi.org/10.29327/243949.2.4-4 Algumas considerações Fazer considerações deste artigo não é algo simples, pois, ele aborda assuntos delicados: Política de Desenvolvimento territorial, Mulheres e a participação social. Constituído de sujeitos que são poucos estudos pela Ciência Geográfica. Ao partir da premissa que por meio das políticas territoriais houve a inserção da mulher no território é de suma relevância compreender as conquistas obtidas e os desafios enfrentando, diante disso, o artigo abordou a importância das ações do território para o fortalecimento da participação das mulheres no TSB. Boletim Alfenense de Geografia v.2, n Boletim Alfenense de Geografia v.2, n.4, 2022. Diagnosticou que é de suma importância quebrar os paradigmas do sistema patriarcal da sociedade brasileira, e que por meio do acesso as políticas públicas territoriais implantadas, as mulheres ganham protagonismo para atuarem nos seus municípios, sejam por meio das reuniões, a exemplo da Câmera Temática de mulheres, ou por meio do suporte para inclusão produtiva, que tem como intuito superar as desigualdades de gênero. nense de Geografia v.2, n.4, 2022. Tendo em vista que há resistência pela emancipação da mulher, é válido ressaltar a importância da participação das mulheres, pois promove a cidadania e autonomia delas dentro das perspectivas das políticas públicas. Portanto, para que haja este fortalecimento é necessário que ocorra uma intensa articulação nos municípios, que tenha uma capacitação e formação continuada acerca dos setores produtivos. Além disso, é de suma importância dar ênfase no suporte financeiro para consolidação e comercialização das unidades produtivas no Território. Nesta quadra, é importante ressaltar o papel do NEDET/UESB no processo de empoderamento dos sujeitos sociais, em especial, os das mulheres por meio das intervenções realizadas nos seus últimos anos. Desta maneira, o texto aponta que o papel da mulher ainda é a base que sustenta a família, por conta disso impede o progresso para que haja mais autonomia nos setores produtivos. Por isso, os desafios enfrentados pelas mulheres nas inclusões produtivas se delineiam a partir da organização dos grupos, pois muitos destes se encontram de forma individualizadas, por conta disso é necessário que obtenha a combinação de estratégias para que aconteça o protagonismo e ao mesmo tempo tenha real orientação para aplicação das políticas pública. Algumas considerações Neste sentindo, a questão do fortalecimento da participação da mulher não é só o estimulo relevante, mas para que ocorra de fato é preciso que tenha uma intensa mobilização por meio do Colegiado Territorial e suas câmaras temáticas buscando o envolvimento de mais entidades que envolva a mulher. E como resultado desta articulação promova um mapeamento destes grupos para que possam ser organizados, além disso, outro fator relevante que pode ser considerado é a falta estrutura e suporte técnico para produção, neste sentindo, para que os grupos possam ser fortalecidos impulsionando a renda no Território. Boletim Alfenense de Geografia v.2, n Boletim Alfenense de Geografia v.2, n.4, 2022. nense de Geografia v.2, n.4, 2022. FERREIRA, Lucas Aguiar Tomaz; ALCANTARA, Fernanda Viana de. A participação social das mulheres no território de identidade sudoeste baiano. Boletim Alfenense de Geografia. Alfenas. v. 2, n.4, p. 53-75, 2022. ISSN: 2764-1422. 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https://openalex.org/W4390980667	https://www.frontiersin.org/articles/10.3389/fneur.2023.1329541/pdf?isPublishedV2=False	English	null	Time-restricted ketogenic diet in amyotrophic lateral sclerosis: a case study	Frontiers in neurology	2,024	cc-by	5,999	OPEN ACCESS OPEN ACCESS EDITED BY Giovanni Meola, University of Milan, Italy REVIEWED BY Dominic D’Agostino, University of South Florida, United States Lucio Comai, University of Southern California, United States CORRESPONDENCE Matthew C. L. Phillips Matthew.Phillips@waikatodhb.health.nz RECEIVED 29 October 2023 ACCEPTED 27 December 2023 PUBLISHED 18 January 2024 CITATION Phillips MCL, Johnston SE, Simpson P, Chang DK, Mather D and Dick RJ (2024) Time-restricted ketogenic diet in amyotroph lateral sclerosis: a case study. Front. Neurol. 14:1329541. doi: 10.3389/fneur.2023.1329541 OPEN ACCESS EDITED BY Giovanni Meola, University of Milan, Italy REVIEWED BY Dominic D’Agostino, University of South Florida, United States Lucio Comai, University of Southern California, United States CORRESPONDENCE Matthew C. L. Phillips Matthew.Phillips@waikatodhb.health.nz RECEIVED 29 October 2023 ACCEPTED 27 December 2023 PUBLISHED 18 January 2024 CITATION Phillips MCL, Johnston SE, Simpson P, Chang DK, Mather D and Dick RJ (2024) Time-restricted ketogenic diet in amyotrophic lateral sclerosis: a case study. Front. Neurol. 14:1329541. doi: 10.3389/fneur.2023.1329541 Matthew C. L. Phillips1*, Samuel E. Johnston2, Pat Simpson3, David K. Chang4, Danielle Mather4 and Rognvald J. Dick2 1Department of Neurology, Waikato Hospital, Hamilton, New Zealand, 2Older Persons and Rehabilitation Service, Waikato Hospital, Hamilton, New Zealand, 3Department of Respiratory Medicine, Waikato Hospital, Hamilton, New Zealand, 4Department of Speech Language Therapy, Waikato Hospital, Hamilton, New Zealand Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder. The most devastating variant is bulbar-onset ALS, which portends a median survival of 24 months from the onset of symptoms. Abundant evidence indicates that neuron metabolism and mitochondrial function are impaired in ALS. Metabolic strategies, particularly fasting and ketogenic diet protocols, alter neuron metabolism and mitochondria function in a manner that may mitigate the symptoms of this disorder. We report the case of a 64-year-old man with a 21-month history of progressive, deteriorating bulbar-onset ALS, with an associated pseudobulbar afect, who implemented a time-restricted ketogenic diet (TRKD) for 18 months. During this time, he improved in ALS-related function (7% improvement from baseline), forced expiratory volume (17% improvement), forced vital capacity (13% improvement), depression (normalized), stress levels (normalized), and quality of life (19% improvement), particularly fatigue (23% improvement). His swallowing impairment and neurocognitive status remained stable. Declines were measured in physical function, maximal inspiratory pressure, and maximal expiratory pressure. Weight loss was attenuated and no signiﬁcant adverse efects occurred. This case study represents the ﬁrst documented occurrence of a patient with ALS managed with either a fasting or ketogenic diet protocol, co-administered as a TRKD. TYPE Case Report PUBLISHED 18 January 2024 DOI 10.3389/fneur.2023.1329541 TYPE Case Report PUBLISHED 18 January 2024 DOI 10.3389/fneur.2023.1329541 motor neuron disease, amyotrophic lateral sclerosis, neurodegeneration, energy metabolism, mitochondria dysfunction, metabolic strategy, fasting, ketogenic diet motor neuron disease, amyotrophic lateral sclerosis, neurodegeneration, energy metabolism, mitochondria dysfunction, metabolic strategy, fasting, ketogenic diet OPEN ACCESS We measured improved or stabilized ALS-related function, forced expiratory volume, forced vital capacity, swallowing, neurocognitive status, mood, and quality of life. Measurable declines were restricted to physical function, maximal inspiratory pressure, and maximal expiratory pressure. Now over 45 months since symptom onset, our patient remains functionally independent and dedicated to his TRKD. COPYRIGHT © 2024 Phillips, Johnston, Simpson, Chang, Mather and Dick. 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. motor neuron disease, amyotrophic lateral sclerosis, neurodegeneration, energy metabolism, mitochondria dysfunction, metabolic strategy, fasting, ketogenic diet Introduction An ALS panel analysis for mutations in 35 diﬀerent genes was negative (ALS Panel, Blueprint Genetics, Espoo, Finland). Blood investigations were normal. At the 3-month follow-up, 21 months after symptom onset, our patient was diagnosed with bulbar-onset ALS with an associated pseudobulbar aﬀect by two independent neurologists (both with neurophysiology fellowships) based on the 2020 Gold Coast criteria (21). Many patients also develop changes in behavior, cognition, and mood. The pathogenesis of ALS involves the degeneration and death of upper motor neurons in the brain motor cortices, lower motor neurons in the brainstem and spinal cord, and neurons in the frontotemporal regions of the brain (4). Approximately 90% of patients lack a family history and are classiﬁed as sporadic ALS, whereas the remaining 10% show a pattern of inheritance associated with a gene mutation and are classiﬁed as familial. Treatment for sporadic and familial ALS is limited and new therapies are needed. Abundant evidence indicates that neuron metabolism and mitochondrial function are impaired in ALS (5–10). On a morphological level, mitochondria are abnormally shaped, swollen, and vacuolated (6, 11). Metabolically, human-derived motor neurons in both sporadic and familial ALS exhibit defective oxidative phosphorylation, ATP loss, and elevated levels of reactive oxygen species (9, 12). Mitochondria in ALS motor neurons also display impaired glucose metabolism, tricarboxylic acid (TCA) cycle activity, calcium buﬀering, axonal transport, and population dynamics (5, 10). Moreover, degradations in cell metabolism and mitochondria function have been documented in astrocytes, microglia, Schwann cells, hepatocytes, lymphocytes, and skeletal muscle cells (6). Collectively, these impairments in cell metabolism and mitochondria function culminate in a chronic bioenergetic challenge that disproportionately impacts metabolically active cells such as neurons, glia, and myocytes. Metabolic strategies, particularly fasting and ketogenic diet protocols, alter cell metabolism and mitochondria function (13). Both strategies enhance production of the dominant blood ketone, beta-hydroxybutyrate (BHB), such that its concentration is sustained at 0.5–0.6 mmol/L or higher (14). BHB metabolism leads to an enhanced free energy of ATP hydrolysis and a greater supply of TCA cycle intermediates (15). Ketone metabolism also produces fewer reactive oxygen species and increases the production of oxidative stress resistance factors (16, 17). Importantly, fasting and ketogenic diet regimens renew the mitochondria pool by upregulating mitogenesis and mitophagy (18). Frontiers in Neurology Introduction Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder that aﬄicts 4.1–8.4 out of every 100,000 people in the world (1), with the number of new cases anticipated to rise over the next 20–25 years (2). Although most patients develop limb-onset ALS, 25–30% of patients develop the more devastating bulbar-onset variant, which is associated with a median survival of 24 months from the onset of symptoms (3). Bulbar-onset ALS typically presents with dysarthria, dysphagia, or dysphonia. 01 Frontiers in Neurology frontiersin.org Phillips et al. 10.3389/fneur.2023.1329541 Phillips et al. 10.3389/fneur.2023.1329541 He was referred to our motor neuron disease clinic, which occurred 9 months later due to resource constraints, by which time his bulbar symptoms had worsened leading to 10 kg of weight loss since symptom onset. Medical history included colorectal cancer (T1N0M0) 12 years previously, which was treated with a hemicolectomy, as well as a myocardial infarction 11 years previously, treated with coronary angioplasty. He had a 2-year history of chronic musculoskeletal pain aﬄicting both shoulders, hips, and ankles. Regular medications included aspirin and metoprolol. He was a life-long smoker (10 cigarettes a day for 50 years—he had quit 4 years previously, but recently resumed smoking due to frustration over his symptoms). There was no family history of neuromuscular disease. Socially, he lived with his wife, who was also his caregiver, and had recently retired from work due to his symptoms. On examination, our patient had a muscular build, with little body fat, and he exhibited inappropriate laughter and crying throughout the consultation. He was 160 cm in height and 72.2 kg in weight, with a calculated body-mass index of 28.2 kg/m2. Neurological examination of the bulbar region revealed deﬁnite tongue wasting and fasciculations, a brisk jaw jerk, and spastic dysarthria. Examination of the cervical region revealed mild wasting of the bilateral supraspinatus and infraspinatus muscles and mild weakness in left shoulder abduction, elbow ﬂexion and extension, and ﬁnger grip and abduction (all 5-/5). All limb reﬂexes were normal (2+) and plantar responses were normal. Electromyography of the bulbar, cervical, thoracic, and lumbosacral regions revealed moderate to severe chronic denervation (reduced recruitment and large- amplitude, long-duration motor unit action potentials) in multiple muscles in all four regions. MRI brain showed mild diﬀuse leukoariosis and MRI spine showed multilevel bilateral foraminal stenoses in the cervical spine, both considered normal for age by a neuroradiologist. Introduction Despite these beneﬁcial eﬀects, current clinical evidence for metabolic strategies in ALS is limited to a handful of studies involving transgenic mouse models—for example, compared with mice maintained on a normal diet, mice sustained on a ketogenic diet show preserved motor performance (19), and mice fed caprylic triglyceride (a medium- chain fatty acid that is readily metabolized into ketones) show a delayed progression of weakness, improved performance, and protection from motor neuron loss (20). Given the deteriorating symptoms, riluzole was oﬀered but our patient declined, after which an 18-month time-restricted ketogenic diet (TRKD) was presented as an option (Figure 1). The TRKD involved reducing feeding times to two meals a day. Our patient chose the timing of the two meals every day and up to 1 h was permitted per meal, ensuring that food intake was limited to 2 h per day, with fasting (allowing only water, tea, and coﬀee) occurring all other hours. The modiﬁed ketogenic diet was roughly 60% fat, 30% protein, 5% ﬁber, and 5% net carbohydrate by weight and comprised primarily of whole foods (green vegetables, meats, eggs, nuts, seeds, creams, and natural oils). Our patient was encouraged to eat to satiation at every meal and not to restrict his calorie intake. After obtaining written informed consent, we provided him with a booklet containing guidelines, recipes, and space to record daily (bedtime) blood glucose and ketone levels using a blood glucose and ketone monitor (CareSens Dual, Pharmaco Diabetes, Auckland, New Zealand). The lead investigator provided support as needed via email. Aside from the TRKD, there were no other Given the collective evidence, we hypothesized that a metabolic strategy might lead to clinical beneﬁts and improved quality of life in a patient with ALS. frontiersin.org Case study We report the case of a 64-year-old male dairy farm systems stock controller of European background who presented to his general practitioner with 9 months of slurring and slowing of speech, diﬃculty swallowing solids, coughing when consuming liquids, intermittent sialorrhea, and constant fatigue. His family had also noted intermittent episodes of laughing and sobbing. 02 frontiersin.org Phillips et al. 10.3389/fneur.2023.1329541 FIGURE 1 Patient timeline. lifestyle changes and our patient continued to smoke 10 cigarettes a day. Delis-Kaplan Executive Function System (28), and the Controlled Oral Word Association Test (COWAT) from the Multilingual Aphasia Examination (29) (for all tests, higher numbers indicated improved neurocognition), as well as mood using the Depression Anxiety Stress Scale (30). Quality of life was reported by the patient and caregiver using the Functional Assessment of Chronic Illness Therapy—Fatigue (FACIT-F) (scores range from 0 to 160, higher numbers indicate better quality of life) (31). Body weight and blood markers were measured at each assessment. We conducted an adverse eﬀects questionnaire with the patient and caregiver at 6, 12, and 18 months. Clinical assessments were conducted during the week preceding the start of the TRKD, with repeat assessments at 6, 12, and 18 months. Clinical assessments evaluated ALS-related function, physical function, pulmonary function, swallowing impairment, neurocognitive status, mood, and quality of life. Assessors were blinded to the intervention. ALS-related function was reported by the patient and caregiver using the Revised ALS Functional Rating Scale (ALSFRS-R) (scores range from 0 to 48, higher numbers indicate improved function) (22). Physical function was measured by a physiotherapist using the get-up-and- go, 6-min walk, and stair climb tests, with an average from two tests calculated for each measure (23–25). Pulmonary function was measured by a pulmonary clinical nurse specialist using spirometry, which assessed forced expiratory volume, forced vital capacity, maximal inspiratory pressure, and maximal expiratory pressure. Swallowing impairment was assessed by speech and language therapists with videoﬂuoroscopic swallowing study and interpreted using the New Zealand Index for Multidisciplinary Evaluation of Swallowing (NZIMES, available at https://ﬂiphtml5. com/nfqi/zyar/basic), which delineates the oral phase, oral transit parameters, pharyngeal phase, crico-esophageal parameters, and laryngeal parameters. Case study Neurocognitive status was measured by a neuropsychologist using the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS, with Form A used at baseline and Week 12 and Form B used at Weeks 6 and 18) (26), Processing Speed from the Wechsler Adult Intelligence Scale—Fourth Edition (27), the Trail Making Test from the During the 18-month TRKD, our patient’s mean blood glucose and ketone levels were 6.52 +/- 0.91 and 0.77 +/- 0.43 mmol/L, respectively (Figure 2), with improvement or stability documented in most outcome measures (Tables 1, 2). Regarding function, the ALSFRS-R improved (42–45, representing a 7% improvement from baseline). The get-up-and-go, 6-min walk, and stair climb tests showed decline. Regarding pulmonary function, the forced expiratory volume improved (2.31–2.70 L, representing a 17% improvement), as did forced vital capacity (3.83–4.33 L, representing a 13% improvement). The maximal inspiratory and expiratory pressures declined. Baseline swallowing impairment remained stable aside from the oral phase (none to mild impairment) and laryngeal parameters (moderate to mild impairment). Despite his stabilized swallowing function, our patient opted for a radiologically inserted gastrostomy (RIG) insertion 9 months into the TRKD to mitigate symptoms of aspiration, which existed before the TRKD was implemented and were only triggered by water (to date, the RIG remains solely 03 Frontiers in Neurology frontiersin.org Phillips et al. 10.3389/fneur.2023.1329541 FIGURE 2 Mean monthly blood glucose and ketone (beta-hydroxybutyrate) levels during the time-restricted ketogenic diet (TRKD). Error bars indicate standard error. FIGURE 2 Mean monthly blood glucose and ketone (beta-hydroxybutyrate) levels during the time-restricted ketogenic diet (TRKD). Error bars indicate standard error. Although fasting and ketogenic diet protocols may individually confer metabolic beneﬁts in ALS, they can be readily combined. Time-restricted feeding eases the burden of organizing multiple meals, which compensates for the extra time required to become familiar with a ketogenic diet, whereas the diet may improve long-term hunger (32), which increases the tolerability of the fasts. The modiﬁed ketogenic diet used here was simple, ﬂexible, palatable, and aﬀordable, which alleviated the restrictions that have been associated with ketogenic diets in the past (33). Regardless of whether metabolic strategies are isolated or combined, it is important to monitor blood glucose and BHB levels so that diﬃculties can be detected and resolved. Case study Despite good adherence to the 18-month TRKD, our patient’s mean blood glucose levels averaged 6.52 mmol/L and his BHB 0.77 mmol/L, which is in the lower range of physiological ketosis (albeit, still within range). This may relate to his body composition and the ALS process itself. Our patient’s lower fat mass probably provided less fuel reserve for fasting-induced ketogenesis. Moreover, many individuals with ALS exhibit skeletal muscle hypermetabolism (5), which may constitute an adaptive response to decreased energy metabolism eﬃciency (34). Hypermetabolism could have increased our patient’s ketone utilization, leading to lower blood levels. used for water intake). The neurocognitive tests generally remained stable. Baseline depression and stress levels resolved (moderate to normal). Regarding quality of life, the FACIT-F improved (114–136, representing a 19% improvement), particularly fatigue as measured by the FACIT-F subscale (35–43, representing a 23% improvement). Our patient’s weight was 72.2 kg at baseline and 69.0 kg at month 18. Blood investigations for hemoglobin, creatinine, liver function tests, and glycosylated hemoglobin remained normal over 18 months. Blood triglycerides increased from 1.3 to 1.6 mmol/L, high-density lipoprotein remained at 1.1 mmol/L, low-density lipoprotein increased from 3.1 to 5.4 mmol/L, and total cholesterol increased from 4.8 to 7.2 mmol/L. Our patient experienced no signiﬁcant adverse eﬀects during the TRKD and consistently mentioned enhanced energy, improved sleep, and reductions in his chronic musculoskeletal pain. Frontiers in Neurology frontiersin.org Discussion p g g g g Outcome Baseline Month 6 Month 12 Month 18 ALS-related function ALSFRS-R Speech 3 3 3 3 Salivation 2 2 2 3 Swallowing 3 3 3 3 Handwriting 4 4 3 4 Handling utensils 4 4 4 4 Dressing and hygiene 4 4 4 4 Turning in bed 4 4 4 4 Walking 4 4 4 4 Climbing stairs 3 4 4 4 Dyspnea 3 4 4 4 Orthopnea 4 4 4 4 Respiratory insuﬃciency 4 4 4 4 Total 42 44 43 45 Physical function Get-up-and-go (s) 6.9 6.6 6.8 7.9 6-min walk (m) 541 568 538 497 Stair climb (s) 8.9 8.3 9.3 10.5 Pulmonary function FEV1 (L, % predicted) 2.31, 86 2.62, 97 2.37, 88 2.70, 99 FVC (L, % predicted) 3.83, 111 3.87, 112 4.14, 120 4.33, 124 MIP (kPa, % predicted) 5.86, 70 4.10, 50 5.00, 60 4.10, 50 MEP (kPa, % predicted) 11.30, 86 12.20, 93 6.70, 51 4.10, 31 Swallowing impairment Oral phase None Mild Mild Mild Oral transit parameters None Mild Mild None Pharyngeal phase Mild Mild Mild Mild Crico-esophageal parameters None None None None Laryngeal parameters Moderate None None Mild ALSFRS-R, Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; FEV1, Forced Expiratory Volume; FVC, Forced Vital Capacity; MIP, Maximal Inspiratory Pressure; MEP, Maximal Expiratory Pressure. ALSFRS-R, Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; FEV1, Forced Expiratory Volume; FVC, Forced Vital Capacity; MIP, Maximal Inspiratory Pressure; MEP, Maximal ALSFRS-R, Revised Amyotrophic Lateral Sclerosis Functional Rating Scale; FEV1, Forced Expiratory Volume; FVC, Forced Vital Capacity; MIP, Maximal Inspiratory Pressure; MEP, Maxima Expiratory Pressure. did not decline over 18 months is potentially important. With respect to pulmonary function, forced vital capacity is arguably the most signiﬁcant spirometric correlate of disease progression in ALS (36), which shows an average decline of 2–3% predicted per month (35). Given our patient’s baseline of 3.83 L (111% predicted), his score should have declined to at least 2.87 L (75% predicted) during the TRKD. Instead, it improved to 4.33 L (124% predicted). Importantly, the maximal inspiratory and expiratory pressures declined during the TRKD, which is discordant with the improved forced expiratory volume and vital capacity, but these measurements should be viewed cautiously as both tests are notoriously diﬃcult to perform and poorly predictive of respiratory capacity (37). Discussion This case study represents the ﬁrst documented occurrence of a patient with ALS managed with either a fasting or ketogenic diet protocol, co-administered as a TRKD. We measured improved ALS-related function (7% improvement from baseline), forced expiratory volume (17% improvement), forced vital capacity (13% improvement), depression (normalized), stress levels (normalized), and quality of life (19% improvement), particularly fatigue (23% improvement). His swallowing impairment and neurocognitive status remained stable. Measurable declines were restricted to physical function, maximal inspiratory pressure, and maximal expiratory pressure. Weight loss was attenuated and no signiﬁcant adverse eﬀects occurred. During this case study, our patient improved or stabilized in most measures of function, which is consistent with TRKD- induced enhancements in neuron, glial cell, and myocyte metabolism as well as mitochondria function. The ALS community relies on the ALSFRS-R to monitor activities of daily living and disease progression (4, 22), which typically declines by 1 point per month (35). Given our patient’s baseline score of 42, his score should have declined to 24 during the TRKD. Instead, it improved to 45. Although ALSFRS-R score changes do not necessarily reﬂect improvement (4), and the speciﬁc tests of physical function declined at the ﬁnal assessment, the fact that our patient’s score Frontiers in Neurology 04 frontiersin.org Phillips et al. 10.3389/fneur.2023.1329541 TABLE 1 Outcome measures for ALS-related, physical, pulmonary, and swallowing function at baseline, 6 months, 12 months, and 18 months after commencing the time-restricted ketogenic diet (for all tests except the get-up-and-go and stair climb, higher numbers indicate improved outcomes; improved or stabilized outcomes are highlighted in blue and declines are highlighted in red). e measures for ALS-related, physical, pulmonary, and swallowing function at baseline, 6 months, 12 months, and 18 months after time-restricted ketogenic diet (for all tests except the get-up-and-go and stair climb, higher numbers indicate improved outcome lized outcomes are highlighted in blue and declines are highlighted in red). Frontiers in Neurology frontiersin.org Discussion Outcome Baseline Month 6 Month 12 Month 18 Neurocognitive status RBANS (scaled score, percentile) Immediate memory 76, 5 76, 5 81, 10 85, 16 Visuospatial/constructional 100, 50 100, 50 105, 63 131, 98 Language 96, 39 96, 39 92, 30 92, 30 Attention 91, 27 82, 12 91, 27 85, 16 Delayed memory 98, 45 100, 50 102, 55 100, 50 Total 88, 21 86, 18 91, 27 97, 42 Processing Speed (scaled score, percentile) 97, 42 86, 18 100, 50 94, 34 Trail Making Test (scaled score, percentile) Visual scanning 12, 75 7, 16 10, 50 10, 50 Number sequencing 14, 91 12, 75 13, 84 11, 63 Letter sequencing 12, 75 12, 75 13, 84 12, 75 Number switching 11, 63 11, 63 10, 50 11, 63 Motor speed 11, 63 10, 50 11, 63 11, 63 COWAT (raw score, percentile) Phonological ﬂuency 18, <10 18, <10 18, <10 17, <10 Semantic ﬂuency 22, 90 21, 90 18, 75 19, 75 Mood Depression Anxiety Stress Scale Depression Moderate Moderate Normal Normal Anxiety Normal Moderate Normal Normal Stress Moderate Moderate Normal Normal Quality of life FACIT-F (overall) 114 135 141 136 FACIT-F subscale (fatigue) 35 43 42 43 RBANS, Repeatable Battery for the Assessment of Neuropsychological Status; COWAT, Controlled Oral Word Association Test; FACIT-F, Functional Assessment of Chronic Illness Therapy - Fatigue. he Assessment of Neuropsychological Status; COWAT, Controlled Oral Word Association Test; FACIT-F, Functional Assessment of Chronic Illness Therapy - During the 21 months prior to the TRKD, our patient lost 10 kg of body weight, which was concerning given that weight loss is negatively correlated with survival in ALS (7, 8). By contrast, he lost only 3.2 kg of weight during the 18-month TRKD. Although the resumption of his smoking habit may have accounted for a portion of the 10 kg of weight loss, the relative weight preservation during the TRKD hints at a weight-sparing eﬀect. It might seem paradoxical that a strategy with the potential to induce a caloric deﬁcit could somehow lead to a weight- sparing eﬀect in ALS. However, the explanation may lie in a TRKD-induced increased eﬃciency of ATP synthesis. ALS involves the accumulation of damaged, uncoupled mitochondria (41), followed by a progressive decrease in energy metabolism eﬃciency, energy dissipation through thermogenesis, and ATP depletion despite failing attempts by hypermetabolism to compensate for the shortfall. Discussion Lastly, swallowing impairment occurs in 85–92% of patients diagnosed with bulbar-onset ALS and is associated with malnutrition, aspiration pneumonia, faster functional decline, and increased mortality (38). Importantly, our patient’s swallowing function remained stable during the 18-month TRKD, with only minor variations noted in the oral phase (slightly declined) and laryngeal parameters (slightly improved), both of which can be attributed to typical variations seen from swallow to swallow. Our patient improved or remained stable in most measures of neurocognitive status, mood, and quality of life. Although ALS is often considered a neuromuscular disorder, 50% of patients exhibit impaired executive function, language ﬂuency, Frontiers in Neurology 05 frontiersin.org Phillips et al. 10.3389/fneur.2023.1329541 TABLE 2 Outcome measures for neurocognitive status, mood, and quality of life at baseline, 6 months, 12 months, and 18 months after commencing the time-restricted ketogenic diet (for all tests, higher numbers indicate improved outcomes; improved or stabilized outcomes are highlighted in blue and declines are highlighted in red). TABLE 2 Outcome measures for neurocognitive status, mood, and quality of life at baseline, 6 months, 12 months, and 18 months after commencing the time-restricted ketogenic diet (for all tests, higher numbers indicate improved outcomes; improved or stabilized outcomes are highlighted in blue and declines are highlighted in red). TABLE 2 Outcome measures for neurocognitive status, mood, and quality of life at baseline, 6 months, 12 months, and 18 months after commencing the time-restricted ketogenic diet (for all tests, higher numbers indicate improved outcomes; improved or stabilized outcomes are highlighted in blue and declines are highlighted in red). frontiersin.org Frontiers in Neurology 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 Fasting may counter this process by inducing mitochondria renewal and coupling, which can increase the rate of ATP synthesis—for example, in a study involving uncoupling or cognition due to pathology in the frontotemporal region (39, 40). The RBANS measures a variety of cognitive domains related to memory, visuospatial and constructional ability, expressive language, and attention (26). Our patient’s low baseline RBANS scores (21st percentile) suggested a degree of baseline executive- type impairment, which remained stable (or improved) after the 18-month TRKD (42nd percentile). The COWAT measures verbal ﬂuency (29). Our patient’s low baseline phonological ﬂuency scores (<10th percentile) indicated substantial language impairment at baseline, which remained stable. The ongoing stability in most of the neurocognitive tests is encouraging and may reﬂect TRKD- associated enhancements in neocortical neuron metabolism and mitochondria function. From the perspective of our patient and his wife, the greatest beneﬁts associated with the TRKD related to his mood and energy levels. His moderate baseline depression and stress levels resolved. He also improved his quality of life and fatigue scores, which allowed him to maintain an active outdoors lifestyle on his farm. Frontiers in Neurology 06 Phillips et al. 10.3389/fneur.2023.1329541 10.3389/fneur.2023.1329541 Funding In conclusion, this case study represents the ﬁrst documented occurrence of a patient with ALS managed with either a fasting or ketogenic diet protocol, co-administered as a TRKD. We measured improved or stabilized ALS-related function, forced expiratory volume, forced vital capacity, swallowing impairment, neurocognitive status, depression, stress levels, and quality of life. Measurable declines were restricted to physical function, maximal inspiratory pressure, and maximal expiratory pressure. Our patient remains functionally independent and dedicated to his TRKD. Despite its limitations, this case study is encouraging and serves as a proof-of-concept for further studies involving metabolic strategies in ALS. The author(s) declare that no ﬁnancial support was received for the research, authorship, and/or publication of this article. 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. Data availability statement The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author. Ethics statement protein-3 knockout mice, fasted mice showed a four-fold higher rate of ATP synthesis compared to fed mice despite no measurable diﬀerence in TCA cycle activity or whole-body energy expenditure (42). The TRKD may have increased ATP synthesis eﬃciency in our patient, culminating in less generated energy being “lost” and a subsequent sparing eﬀect on muscle and fat reserves. Consistent with this hypothesis, well-designed trials have shown that simply increasing calorie intake, which would not increase metabolic eﬃciency, does not signiﬁcantly attenuate weight loss in people with ALS (43, 44). Ethical approval was not required for this study involving a human participant because local and institutional review boards do not require ethics approval for case reports/studies, so long as written informed consent is obtained. The study was conducted in accordance with the local legislation and institutional requirements. The participant provided written informed consent to participate in this study. Written informed consent was obtained from the individual for the publication of any potentially identiﬁable images or data included in this article. Given that this is a case study, we cannot draw ﬁrm conclusions regarding the mechanism of the documented improvements or potential impact of the TRKD on survival. Clinical features associated with shorter survival times include bulbar-onset ALS, older age, rapid functional decline (as measured by the ALSFRS- R), low forced vital capacity, frontotemporal dementia, and pronounced weight loss (3). Given that most of these negative prognostic features either improved or stabilized in our patient, it is reasonable to anticipate an ongoing TRKD-induced survival beneﬁt. Alternative explanations for some of the improvements include a practice eﬀect and a placebo eﬀect. 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https://openalex.org/W2795373842	https://hal.umontpellier.fr/hal-01957306/document	English	null	Comparative Epidemiology of Highly Pathogenic Avian Influenza Virus H5N1 and H5N6 in Vietnamese Live Bird Markets: Spatiotemporal Patterns of Distribution and Risk Factors	Frontiers in veterinary science	2,018	cc-by	10,224	To cite this version: Kate C Mellor, Anne Meyer, Doaa A Elkholly, Pham T Long, Guillaume Fournié, et al.. Comparative Epidemiology of Highly Pathogenic Avian Influenza Virus H5N1 and H5N6 in Vietnamese Live Bird Markets: Spatiotemporal Patterns of Distribution and Risk Factors. Frontiers in Veterinary Science, 2018, 5, pp.51. 10.3389/fvets.2018.00051. hal-01957306 Distributed under a Creative Commons Attribution 4.0 International License Abbreviations: HPAI, highly pathogenic avian influenza; LBM, live bird market; INLA, integrated nested Laplace approxima- tions; LISA, local indicators of spatial association; DAH, Department of Animal Health; CAR, conditional autoregressive model. HAL Id: hal-01957306 https://hal.umontpellier.fr/hal-01957306v1 Submitted on 17 Dec 2018 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Original Research published: 05 April 2018 doi: 10.3389/fvets.2018.00051 Comparative Epidemiology of Highly Pathogenic Avian Influenza Virus H5N1 and H5N6 in Vietnamese Live Bird Markets: Spatiotemporal Patterns of Distribution and Risk Factors Kate C. Mellor1, Anne Meyer1, Doaa A. Elkholly1, Guillaume Fournié1, Pham T. Long2, Ken Inui3, Pawin Padungtod3, Marius Gilbert4, Scott H. Newman3,5†, Timothée Vergne1,6,7, Dirk U. Pfeiffer1,8 and Kim B. Stevens1 Keywords: avian influenza, epidemiology, live bird markets, poultry, spatial modelling, Vietnam, emerging infectious disease Edited by: Moh A. Alkhamis, Kuwait University, Kuwait Reviewed by: Marta Hernandez-Jover, Charles Sturt University, Australia Luis Gustavo Corbellini, Federal University of Rio Grande do Sul (UFRGS), Brazil Edited by: Moh A. Alkhamis, Kuwait University, Kuwait 1 Veterinary Epidemiology, Economics and Public Health Group, Department of Pathobiology and Population Sciences, Royal Veterinary College, Hatfield, United Kingdom, 2 Department of Animal Health, Ministry of Agriculture and Rural Development, Hanoi, Vietnam, 3 Country Office for Vietnam, Food and Agriculture Organization of the United Nations, Hanoi, Vietnam, 4 Spatial Epidemiology Laboratory, Université Libre de Bruxelles, Brussels, Belgium, 5 Country Office for Ethiopia, Food and Agriculture Organization of the United Nations, Addis Ababa, Ethiopia, 6 Maladies Infectieuses et Vecteurs Ecologie, Génétique, Evolution et Contrôle (MIVEGEC), Institut de Recherche pour le Développement (IRD), Montpellier, France, 7 UMR 1225 INRA, ENVT Interactions Hôtes – Agents Pathogènes (IHAP), University of Toulouse, Toulouse, France, 8 College of Veterinary Medicine & Life Sciences, City University of Hong Kong, Kowloon, Hong Kong Reviewed by: Marta Hernandez-Jover, Charles Sturt University, Australia Luis Gustavo Corbellini, Federal University of Rio Grande do Sul (UFRGS), Brazil Correspondence: Kate C. Mellor kmellor5@rvc.ac.uk Highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Vietnam since 2003, whilst outbreaks of HPAI H5N6 virus are more recent, having only been reported since 2014. Although the spatial distribution of H5N1 outbreaks and risk factors for virus occurrence has been extensively studied, there have been no comparative studies for H5N6. Data collected through active surveillance of Vietnamese live bird markets (LBMs) between 2011 and 2015 were used to explore and compare the spatiotemporal distribu- tions of H5N1- and H5N6-positive LBMs. Conditional autoregressive models were devel- oped to quantify spatiotemporal associations between agroecological factors and the two HPAI strains using the same set of predictor variables. Unlike H5N1, which exhibited a strong north–south divide, with repeated occurrence in the extreme south of a cluster of high-risk provinces, H5N6 was homogeneously distributed throughout Vietnam. Similarly, different agroecological factors were associated with each strain. Sample collection in the months of January and February and higher average maximum temperature were associated with higher likelihood of H5N1-positive market-day status. The likelihood of market days being positive for H5N6 increased with decreased river density, and with successive Rounds of data collection. This study highlights marked differences in spatial patterns and risk factors for H5N1 and H5N6 in Vietnam, suggesting the need for tailored surveillance and control approaches. Highly pathogenic avian influenza (HPAI) H5N1 virus has been circulating in Vietnam since 2003, whilst outbreaks of HPAI H5N6 virus are more recent, having only been reported since 2014. Although the spatial distribution of H5N1 outbreaks and risk factors for virus occurrence has been extensively studied, there have been no comparative studies for H5N6. Data collected through active surveillance of Vietnamese live bird markets (LBMs) between 2011 and 2015 were used to explore and compare the spatiotemporal distribu- tions of H5N1- and H5N6-positive LBMs. Conditional autoregressive models were devel- oped to quantify spatiotemporal associations between agroecological factors and the two HPAI strains using the same set of predictor variables. Unlike H5N1, which exhibited a strong north–south divide, with repeated occurrence in the extreme south of a cluster of high-risk provinces, H5N6 was homogeneously distributed throughout Vietnam. Similarly, different agroecological factors were associated with each strain. Sample collection in the months of January and February and higher average maximum temperature were associated with higher likelihood of H5N1-positive market-day status. The likelihood of market days being positive for H5N6 increased with decreased river density, and with successive Rounds of data collection. This study highlights marked differences in spatial patterns and risk factors for H5N1 and H5N6 in Vietnam, suggesting the need for tailored surveillance and control approaches. †Present address Scott Newman, Regional Office for Africa, Senior Animal Health & Production Officer, Food and Agriculture Organization of the United Nations, Accra, Ghana †Present address Scott Newman, Regional Office for Africa, Senior Animal Health & Production Officer, Food and Agriculture Organization of the United Nations, Accra, Ghana Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Received: 30 November 2017 Accepted: 27 February 2018 Published: 05 April 2018 Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Specialty section: This article was submitted to Veterinary Epidemiology and Economics, a section of the journal Frontiers in Veterinary Science Received: 30 November 2017 Accepted: 27 February 2018 Published: 05 April 2018 Surveillance Characteristics Surveillance Protocol Sampling was conducted as part of routine governmental active surveillance. All surveillance activities and protocols were approved by the Vietnamese Department of Animal Health (DAH) Epidemiology Division before implementation. Sampling activities were implemented at specified times and places by the provincial Sub-Department of Animal Health (SDAH). At the LBM level, a sample size of 30 was required for 95% confidence of detection of H5N1 or H5N6, assuming prevalence of 10%, test sensitivity of 90%, and specificity of 99%. Sample testing was conducted in seven Regional Animal Health Office BSL2+ certified laboratories belonging to DAH, using BSL3 biosafety practice. In response to the endemic HPAI status in Vietnam, extensive active surveillance of live bird markets (LBMs) was initiated in 2008, first targeting the H5N1 virus and later expanding to include the H5N6 strain. LBMs were chosen as the foci for sur- veillance activities, partly because funding constraints precluded active surveillance at the farm level, but also because LBMs act as potential reservoirs for HPAI due to their role as hubs for poul- try trade (10–12). Moreover, LBMs in northern Vietnam have been found to be highly connected through contact networks, enabling spread of HPAI not only between markets, but also between regions and even across international borders (12, 13). The key role that LBMs play in endemic spread of the virus was highlighted by the impact of the introduction of various biose curity measures and infection control policies. Requirements such as the introduction of a day of market closure, cleaning at regular intervals, and for all birds to be sold or slaughtered by the end of trading each day greatly reduced the prevalence of HPAI in Hong Kong LBMs (14).h The surveillance period extended from September 2011 to December 2015 and was divided into six “Rounds” (Table 1). INTRODUCTION predictor variables. The majority of studies investigating HPAI in Asia utilise passive surveillance data, which relies upon detection and testing of clinically affected birds. Whilst HPAI H5N1 has been detected in asymptomatic ducks and poultry in LBM (15, 16), such cases are not detected through passive surveillance. The data collected through active surveillance of Vietnamese LBMs over a 5-year period provide a unique opportunity to explore HPAI epidemiology in Vietnam using virus detection data that are less exposed to reporting bias compared with data from passive surveillance. Specific objectives of this study were to (i) determine the prevalence of H5N1 and H5N6 virus in Vietnamese LBMs between 2011 and 2015; (ii) explore the spatiotemporal distribu- tions of H5N1 and H5N6 virus in Vietnam; and (iii) develop models to quantify the spatiotemporal association between agroecological factors and the two HPAI strains using the same set of predictor variables. Highly pathogenic avian influenza (HPAI) H5N1 virus (here- after H5N1) is endemic to multiple Asian countries, including Vietnam, where the first recorded H5N1 outbreak occurred in 2003. Since then, costly control measures have been introduced, including culling of infected birds and vaccination of poultry (1). In addition to the economic impact, H5N1 has a high mortality rate in humans coupled with an ever-present threat of pandemic influenza (2). HPAI H5N6 virus (hereafter H5N6) emerged in Vietnam in April 2014 (3, 4). Both virus subtypes are highly pathogenic in chickens, may cause asymptomatic infection in ducks, and have been associated with sporadic human infection and deaths in Asia. Numerous studies have explored the epidemiology of H5N1 in Asia, describing its spatial distribution at the regional level and in indi- vidual countries. Multiple factors have been associated with H5N1 including increased density of domestic ducks (5, 6) and chickens (7), proximity to high aggregations of human population density, a greater percentage of land used as rice paddy fields, higher rice-cropping intensity and lower average annual precipitation (8, 9). These studies predominantly analysed passive surveillance disease presence data resulting in exposure to temporal and spatial variations in surveillance effectiveness. However, contrast to the extensive literature surrounding H5N1, little has been published on the epidemiology of H5N6 in poultry. Citation: Mellor KC, Meyer A, Elkholly DA, Fournié G, Long PT, Inui K, Padungtod P, Gilbert M, Newman SH, Vergne T, Pfeiffer DU and Stevens KB (2018) Comparative Epidemiology of Highly Pathogenic Avian Influenza Virus H5N1 and H5N6 in Vietnamese Live Bird Markets: Spatiotemporal Patterns of Distribution and Risk Factors. Front. Vet. Sci. 5:51. doi: 10.3389/fvets.2018.00051 April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 1 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. LBM Selection Selection of sampling locations varied by Round as follows: Selection of sampling locations varied by Round as follows: 1. Round 1: samples were collected from the two largest LBMs in each of 30 provinces out of a total of 63 (58 provinces and 5 centrally controlled municipalities (cities) at the same level as provinces). Provinces were selected on the basis of fulfilling one or more of the following criteria: (i) having a previous history of HPAI outbreaks, (ii) presence of an international 1. Round 1: samples were collected from the two largest LBMs in each of 30 provinces out of a total of 63 (58 provinces and 5 centrally controlled municipalities (cities) at the same level as provinces). Provinces were selected on the basis of fulfilling one or more of the following criteria: (i) having a previous history of HPAI outbreaks, (ii) presence of an international The aim of this study was to analyse the spatial distribution of H5N1 and H5N6 in Vietnamese LBMs using the same set of Table 1 \| Graphical illustration of the temporal distribution of the six rounds of active surveillance sampling between September 2011 and December 2015. January February March April May June July August September October November December 2011 Round 1 2012 Round 1 (cont) Round 2 2013 Round 2 (cont) Round 3 2014 Round 3 (cont) Round 4 Round 5 2015 Round 6 2 Frontiers in Veterinary Science \| www.frontiersin.org April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org H5N1 and H5N6 Epidemiology Vietnam Mellor et al. border, (iii) having a high density of poultry, or (iv) high human population density. border, (iii) having a high density of poultry, or (iv) high human population density. swabs from chickens were collected during the last Round only. Environmental sampling started during Round 2 with the col- lection of faeces, feathers and waste in the resting and slaughter areas at four selected large LBMs. Four samples of each type were collected and tested individually. From Round 5 onwards, environmental sampling was extended to all LBMs regardless of their size and environmental swabs were pooled instead of being tested individually. Pooled samples comprised five merged swab samples (either oropharyngeal or environmental). Depending on the Round, between 93 and 100% of the market days reached the sample size targets (detailed in Table 2) for each type of sample. 2. Agroecological Predictor Data A review of the published literature served to identify potential predictor variables previously shown to be risk factors for HPAI occurrence and the final set of predictor variables used in this study included the following: density of ducks (heads/km2) (7, 8, 17, 18), density of chickens (heads/km2) (7, 8, 18, 19), human population density (heads/km2) (6–8, 18, 20), travel time (minutes) to the nearest city with a population of ≥50,000, suitability of areas for growing rice (8, 18), river density (km length/km2) (7, 19, 21, 22), average annual precipitation (mm) (23), average monthly minimum temperature (°C), and average monthly maximum temperature (°C) (23–25). LBM density (number of LBM/10 km2) was also included. LBM Selection Rounds 2–5: samples were collected from provinces distributed throughout Vietnam using the same criteria for province selection as in Round 1. For every round, the DAH selected (i) 40 provinces from which one small-scale LBM was sampled and (ii) 20 provinces from which a large-scale LBM was sam- pled. The DAH selected 120 districts from the aforementioned 40 provinces (three districts per province) and 20 cities from the aforementioned 20 provinces. Districts were selected on the basis of (i) having a high duck den- sity and (ii) having a history of H5N1 outbreaks. In each selected district or city, the SDAH of the corresponding province selected one LBM for sampling. LBMs were selected on the basis of (i) size (at least six vendors), (ii) source of birds (within the district for small-scale LBMs, outside the province for large-scale LBMs), and (iii) no inclusion in H7N9 surveillance activities.i Case Definition Samples were tested at Regional Animal Health Offices for the H5 and N1 virus subtypes using real-time reverse transcription polymerase chain reaction. From Round 4 onwards samples were also tested for the N6 subtype. Cycle threshold values of less than 35 were regarded as positive. Samples positive for both the H5 and N1 subtypes were classified as positive for H5N1. Similarly, samples positive for both the H5 and N6 subtypes were classified as positive for H5N6.h Small-scale markets were defined as markets which draw birds from within the same district and/or province. Sampling should therefore capture/represent the local circulation of HPAI. Large- scale markets were defined as markets which draw birds from outside the province and therefore capture/represent the national and/or regional circulation of HPAI. The epidemiological unit for this study was a market day at a given LBM on a given date. A market day was classified as positive for H5N1 or H5N6 if one or more samples (individual or pooled) collected from that LBM on that date tested positive. 3. Round 6: DAH selected 32 provinces distributed throughout the country; 12 northern provinces that share a border with China or have poultry trading connected to northern border provinces, and 20 central/southern provinces. In the northern provinces, a total of 48 LBM (4 from each province) were sampled. In the central/southern provinces, the largest LBM in each province was sampled. April 2018 \| Volume 5 \| Article 51 Data Collection Sampling On the day of sampling, the vendors to be sampled were selected randomly from all vendors present at the market selling more than five ducks (or chickens for Round 6). The number and type of samples collected for each market day according to the surveil- lance design is summarised in Table 2. Digital spatial data layers representing each predictor variable were sourced for Vietnam from the public domain, and all spatial Oropharyngeal swabs from ducks were collected consist- ently during each surveillance Round, whilst oropharyngeal Table 2 \| Target number of samples to be collected per market day, according to round and sample type. Sample type Numbers refer to pooled samples when not indicated otherwise Round 1 Rounds 2–4 Round 5 Round 6 Oropharyngeal swabs Ducks 4 6 6 6 Chicken 0 0 0 6 Environmental swabs from four large live bird markets Faeces from cage 0 4 individual samples 0 0 Waste from resting area 0 4 individual samples 0 0 Feathers 0 4 individual samples 0 0 Dirt in slaughter area 0 4 individual samples 0 0 Environmental swabs from all sampling sites Liquid waste 0 0 2 2 Solid waste 0 0 2 2 Faeces 0 0 1 1 Drinking water 0 0 1 1 Pooled samples are the combination of 5 swab samples. 3 Frontiers in Veterinary Science \| www.frontiersin.org April 2018 \| Volume 5 \| Article 51 April 2018 \| Volume 5 \| Article 51 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. data manipulations and map creation were performed using ArcGIS 10.3.1 (28). Chicken and duck densities were extracted from the Gridded Livestock of the World (resolution: 1 km2),1 and human population density was obtained from Gridded Population of the World v4 (resolution: 1 km2; estimated for 2015).2 The predicted density of LBMs/10 km2 was obtained from a model generated by Gilbert et al. (unpublished, model description in Supplementary Material; resolution: 10 km2), which was resampled to a resolu- tion of 1 km2. Travel time to the nearest city was obtained from the Global Environment Monitoring Unit in the Joint Research Centre of the European Commission (26). Data Collection Sampling Areas suitable for rice growing were extracted from Suitability for Rain-fed and Irrigated Rice (High Input), a shapefile available from Food and Agricultural Organization’s GeoNetwork website (published 2007).3 The data were converted to raster format (resolution: 1 km2), and the original eight suitability categories were recategorised as follows: high (very high/high/good), moderate (medium/moderate), low (marginal/very marginal), unsuitable. Open water features were extracted from VMap0 Perennial Water Courses (Rivers) of the World (published 1997) (available from the Food and Agricultural Organization’s GeoNetwork website; see text footnote 3) and density of rivers per square kilometre calculated using the line density feature in ArcGIS. Average monthly precipitation and minimum and maximum temperature data (based on the time frame 1950–2000) were obtained from the WorldClim website ((27); accessed March 2017). A vector shapefile of Vietnam’s provincial boundaries was obtained from the GADM Database of Global Administrative Areas v2.8.4 All data were processed to ensure that projections and extents matched. Latitude and longi- tude were available for LBMs, and data for each predictor variable were extracted to the point location of individual LBMs. calculated for each Round as the number of positive market days in a province divided by the total number of market days sampled in that province. Choropleth maps of raw rates or standard mortality ratios per area can be misleading; the addition of a small number of cases in an area with a small population at risk can dramatically increase the reported rate of disease for the area. Conversely, the addition of the same number of cases in an area with a large population at risk has little effect on the reported rate of disease for the area. Bayesian approaches allow disease rates to be adjusted through combining the observed rate for an area with rates observed in surrounding areas. When the at risk population of an area of interest is large, and the statistical error of the rate estimate small, higher credibility is given to the observed estimate, and the Bayes adjusted rates are similar to observed rates. However, where the population at risk is small, the rate is adjusted towards the mean rate observed over the wider study area. 1 https://livestock.geo-wiki.org/home-2/ (Accessed: March, 2017). 2 http://sedac.ciesin.columbia.edu/data/collection/gpw-v4 (Accessed: March, 2017). 3 http://www.fao.org/geonetwork/srv/en/main.home (Accessed: March, 2017). 4 http://www.gadm.org/ (Accessed: March, 2017). Data Collection Sampling Choropleth maps of empirical Bayes-smoothed prevalence were generated for H5N1 and H5N6 using Eqs 1–3 as follows: given that yi equalled the number of positive market days observed in the ith province, ni the total number of market days sampled in the ith province, and ri was the proportion of positive market days for the ith province, then the pooled mean of observed prevalence across all provinces (γ) was calculated as follows: γ =∑ y n i i , (1) (1) and the estimate of the population variance of the prevalence based on a weighted sample of the observed prevalences (φ) was calculated as follows: ϕ γ γ = − ( ) − ∑ ∑ n r n n i i i 2 , (2) (2) Statistical Analysis Data Managementh then θ, the empirical Bayes-smoothed prevalence for the ith province, was calculated as follows: The Regional Animal Health Offices used Microsoft Excel to com- pile the sample collection spreadsheet and laboratory results into a single dataset (regional dataset). These regional datasets were submitted to the DAH each month where they were aggregated to provide a single dataset for each surveillance period. However, merged datasets were not available for some periods, and data recording was not harmonised between regional datasets result- ing in multiple names identifying the same location. In such instances, markets with different names but the same coordinates were considered to be the same market. Eighty-three LBMs with missing longitude and latitude data were assigned the coordinates of the relevant commune centroid. θ ϕ γ ϕ γ γ = ∗ − ( ) + + r n i i . (3) (3) Exploring Spatial Autocorrelation and Clustering Exploring Spatial Autocorrelation and Clustering Spatial autocorrelation of the smoothed Bayes risk was explored at a global scale using the Moran’s I statistic and at a local scale using the Anselin Local Moran’s I statistic and Getis-Ord GI* statistic. The global Moran’s I statistic was used to assess the presence, strength and direction of spatial autocorrelation over the whole study area, using a queen’s contiguity weights matrix and 499 random permutations. A p-value ≤ 0.05 was considered significant. The Local Moran’s I and GI* statistics were used to detect clustering of provinces with similar risk of H5N1 or H5N6, and to identify the locations of province-level hot and/or cold spots. The GI* statistic returned a z-score for each province and for statistically significant positive z-scores, the larger the z-score the more intense the clustering of high values (hot spot). For statistically significant negative z-scores, the smaller the z-score the more intense the clustering of low values Mapping Spatiotemporal Distribution Mapping Spatiotemporal Distribution To preserve LBM anonymity, market days were aggregated by province, and province-level prevalence of H5N1 and H5N6 was To preserve LBM anonymity, market days were aggregated by province, and province-level prevalence of H5N1 and H5N6 was April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 4 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. (cold spot). All spatial analyses were conducted using tools pro vided in ArcGIS 10.3.1 (28). varying random effect “Province,” using a spatial weights matrix where polygons were classified as neighbouring if they shared a corner or border (queen’s contiguity). Clustering of market days within markets was accounted for through the inclusion of the non-spatially varying random effect “Market.” The potential tem- poral effect of sampling heterogeneity was accounted for through inclusion of the variable “Round” in the model. Modelling Associations Between Agroecological Factors and HPAI Multivariable logistic regression analyses were used to inves- tigate associations between putative predictor variables and H5N1 or H5N6-positive market days. Univariable analyses for each predictor variable were conducted, with significant variables included in multivariable analysis. All univariable and multivariable statistical analyses were performed in R 3.4.0 (29). Before multivariable analysis, all predictor data were standardised to a mean of 0 and SD of 1, for variables measured at different scales to contribute equally to the analysis. To identify the set of predictors associated with H5N1 and H5N6- positive market days, non-spatial generalised linear models were used, implemented via the R glmulti package (30), to build every possible non-redundant model for every combination of predictor main effects (interactions were not included due to the number of variables involved). Final best-fit models were chosen using Akaike’s information criterion (AIC), which ranks models based on goodness-of-fit and complexity, whilst penal- ising deviance. The predictors identified in this first step were then included in a mixed-effects logistic regression model with the variable “market” as a random effect to determine whether any predictors were no longer significant after accounting for non-independence of market days. All continuous variables were assessed for linear trend by comparing the model with the continuous version of the variable with a model where the variable was categorised into quartiles. If the likelihood ratio test p-value was <0.05 the categorical version of the variable was included in model.ii All CAR models were implemented in R using integrated nested Laplace approximations (INLA) which uses an approxi mation for inference and avoids the computational demands, convergence issues and mixing problems sometimes encoun- tered by Markov chain Monte Carlo algorithms (31). The model was fitted using R-INLA, with the Besag model for spatial effects specified inside the function. In the Besag model, Gaussian Markov random fields are used as priors to model spatial dependency structures and unobserved effects. In addition, each model was run through INLA whilst excluding the random spatial effect to obtain non-spatial Bayesian estimates and to compare model fit and performance due to the explicit spatial process. Model selection was based on the deviance information criterion (DIC) where a lower DIC indicates a better model fit. In all analyses, an α-level of 0.05 was adopted to indicate statistical significance. Modelling Associations Between Agroecological Factors and HPAI Choropleth maps showing the spatial distribution of the pos- terior means of the structured random effects obtained from the models were produced in ArcGIS (28). April 2018 \| Volume 5 \| Article 51 Sampling Sites and Samples p g p During the surveillance period 22,185 pooled samples were collected from 459 LBM distributed between 48 provinces (242 districts) (Table 3). Each LBM was visited between 1 and 28 times (median 4 visits), providing a total of 3,461 market days for analysis. Sampling intensity was highest in Round 1 and decreased thereafter (Table 3). All identified predictors which remained significant at the 5% level in the mixed-effects logistic regressions were then included in a conditional autoregressive model (CAR) to account for the spatial autocorrelation of observations. Clustering of markets within provinces was accounted for by the inclusion of a spatially Table 3 \| Sampling characteristics and prevalence of highly pathogenic avian influenza H5N1- and H5N6-positive market days of the six surveillance Rounds. Rounds 1 2 3 4 5 6 Total Dates September 2011–February 2012 October 2012–September 2013 October 2013–April 2014 April 2014–October 2014 November 2014–December 2014 July 2015–December 2015 September 2011–December 2015 Number of pooled samples 3,952 4,642 3,984 5,301 1,668 2,638 22,185 Number of provinces 30 44 42 44 44 30 48 Number of districts 122 141 135 138 71 58 242 Number of live bird markets 279 152 143 143 77 63 459 Number of days 153 365 212 184 61 183 1,158 Total market days 974 748 624 827 142 146 3,461 Sampling intensity (market days/number of days) 6.4 2.1 3.0 4.5 2.3 0.8 3.0 Observed prevalence H5N1-positive market days (%) 8.5 (41/974) 19.5 (146/748) 15.7 (111/624) 6.8 (56/827) 14.5 (18/142) 10.2 (15/146) 11.2 (387/3,461) Observed prevalence H5N6-positive market days (%) 0.7 (6/827) 16.2 (23/142) 26.0 (38/146) 6.01 (67/1,115) d prevalence of highly pathogenic avian influenza H5N1- and H5N6-positive market days of the six surveillance Rounds. April 2018 \| Volume 5 \| Article 51 April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 5 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. although the level of risk increased between Rounds 4 and 6 (Figure 4). In general, the most common pattern was one of outliers; provinces with a high risk of H5N6-positive market days were generally surrounded by low-risk provinces and vice versa (Figure 5). Two hot-spot provinces were identified in each Round although Quang Ngai was the only province to be identified as a hot spot more than once (Rounds 4 and 5; Figure 6). Prevalence of H5N1 and H5N6-Positive Market Daysh The observed prevalence of H5N1-positive market days varied between rounds (median: 12.35; range: 6.8–19.5%) although this difference was not significant (χ2 p-value = 0.48) (Table 3). The observed prevalence of H5N6-positive market days increased significantly (χ2 p < 0.001) over Rounds 4–6 from 0.7 to 26% (Table 3). Risk Factors for H5N1 and H5N6-Positive Market Daysh The most robust H5N1 multivariable model, based on the AIC, included six of the thirteen predictor variables; suitability for rice- growing, sampling month, average monthly maximum tempera- ture, river density, travel time to a city and chicken density, and were therefore included in the H5N1 INLA model. The variable “Round” was forced into the model to account for temporal vari- ation in sampling. The CAR model based on these variables had a DIC value of 1,984.31 (H5N1). Inclusion of the spatial random effect “province” improved the fit of the H5N1 model by 8.17%, reducing the DIC to, 1,822.10. Three of the six variables retained in the model were statistically significant, three variables were not deemed significant, due to the odds ratio (OR) 95% credible interval crossing 1. The odds of a market day being positive for H5N1 varied between Rounds. Comparison of OR across months identified the likelihood of market day status being positive to be highest in January and February. The odds of a market day being positive for H5N1 were 3.36 (95% CrI 1.29, 8.36) greater where the average maximum temperature was ≥30.33°C compared with areas where the average maximum temperature was ≤24.47°C (Table 4). Sampling Sites and Samples In general, sampled provinces were evenly distributed through- out the country although sampling in Rounds 2–4 provided more homogenous coverage of Vietnam than Rounds 1 and 6, with the latter exhibiting a slight north–south emphasis. Spatiotemporal Distribution of H5N1- and H5N6-Positive Market Days Figure 2 \| Local Indicators of Spatial Association cluster maps and Moran’s I statistics of highly pathogenic avian influenza H5N1 Bayes risk for Rounds 1–6. Figure 2 \| Local Indicators of Spatial Association cluster maps and Moran’s I statistics of highly pathogenic avian influenza Figure 3 \| Getis-Ord GI* statistic maps showing hot-spot provinces for highly pathogenic avian influenza H5N1 Bayes risk for Rounds 1–6. Figure 3 \| Getis-Ord GI* statistic maps showing hot-spot provinces for highly pathogenic avian influenza H5N1 Bayes risk for Rounds 1–6. etis-Ord GI* statistic maps showing hot-spot provinces for highly pathogenic avian influenza H5N1 Bayes risk for Rounds 1–6. the southern and central provinces of Vietnam. Similarly, whilst higher risk of H5N6-positive LBMs was associated with lower river density, spatial variation in H5N1 risk was primarily associ- ated with climatic factors. was lowered by 0.03% to 202.29). Therefore, the final model used for H5N6 therefore did not include the spatially varying random effect. The likelihood of a market day being positive for H5N6 was higher with successive Rounds and lower river density (Table 5). Mapping posterior means of the spatially structured random effects for H5N1 showed them to be reasonably homogenously distributed throughout the country, suggesting that there is unexplained variation in most regions, after accounting for the model covariates (Figure 7). was lowered by 0.03% to 202.29). Therefore, the final model used for H5N6 therefore did not include the spatially varying random effect. The likelihood of a market day being positive for H5N6 was higher with successive Rounds and lower river density (Table 5). Mapping posterior means of the spatially structured random effects for H5N1 showed them to be reasonably homogenously distributed throughout the country, suggesting that there is unexplained variation in most regions, after accounting for the model covariates (Figure 7). Collection month was associated with variation in market-day H5N1 risk. The odds of H5N1-positive market days were highest in January and February. No samples collected between January and March were tested for H5N6. Seasonal fluctuations in the proportion of positive market days may be due to a combination of climatic factors and peaks in demand for poultry products. Higher incidence of H5N1 in domestic poultry in central and southern Vietnam has been shown to coincide with an increased demand for poultry products in January and February associated with the Lunar New Year Festival (32). Spatiotemporal Distribution of H5N1- and H5N6-Positive Market Days Colder temperatures in winter months have also been proposed to contribute to higher risk of H5N1 due to longer virus survival time in the environment (24, 25). Spatiotemporal Distribution of H5N1- and H5N6-Positive Market Days y Province-level Bayes-smoothed prevalence of H5N1-positive market days was spatially heterogeneous in all six Rounds, although it was highest in Rounds 2 and 6 (Figure 1). This appar- ent spatial heterogeneity was supported by both the global and local autocorrelation statistics, which identified significant posi- tive spatial autocorrelation in all rounds except Round 2 (Moran’s I p-value > 0.05; Figure 2). The positive autocorrelation was characterised by repeated occurrence, in the south of the country, of a cluster of high-risk provinces surrounded by other high-risk provinces, although the size of the cluster varied between Rounds (Figure 2). Conversely, northern Vietnam was characterised by low-risk provinces surrounded by other provinces of low risk. However, the north also exhibited periodic recurrent outliers; provinces with a high disease risk but surrounded by others with a low disease risk (Figure 2). Hot-spot provinces were identified in all Rounds but the number decreased over time (Rounds 1–3: n = 4; Rounds 4–5: n = 2; Round 6: n = 1) (Figure 3). One province, in particular, Ca Mau was identified as a hot-spot province of H5N1-positive market days in four of the six rounds. Unlike H5N1, province-level empirical Bayes-smoothed risk of H5N6-positive market days did not display any significant spatial heterogeneity in any of the Rounds (Moran’s I p-value > 0.05) In the multivariable analysis, only three of the thirteen predic- tors were significantly associated with market days being positive for H5N6: river density, human population density and market density. These covariates were taken forward to the H5N6 INLA model. The variable “Round” was forced into the model to account for temporal variation in the sampling. The CAR model based on these variables had a DIC value of 202.36, inclusion of the spatial random effect “province” did not improve the model fit (the DIC Figure 1 \| Province-level Bayes risk of highly pathogenic avian influenza H5N1 in Vietnam (Rounds 1–6). Figure 1 \| Province-level Bayes risk of highly pathogenic avian influenza H5N1 in Vietnam (Rounds 1–6). April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 6 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. Figure 2 \| Local Indicators of Spatial Association cluster maps and Moran’s I statistics of highly pathogenic avian influenza H5N1 Bayes risk for Rounds 1–6. Frontiers in Veterinary Science \| www.frontiersin.org DISCUSSION The results of this study suggest that the epidemiology of H5N1 and H5N6 in Vietnam are very different. Not only do the two strains show different distributions, they are also associated with different risk factors. Whilst the risk of H5N6-positive market days was homogenous across Vietnam, the posterior mean probabilities of H5N1 from the CAR model at the province level showed clear regional differences, with higher probabilities in Higher average maximum temperature was associated with higher risk of market day H5N1 positivity. This factor contributes to the observed north/south risk divide, as average maximum April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 7 Mellor et al. H5N1 and H5N6 Epidemiology Vietnam Figure 4 \| Province-level empirical Bayes risk of highly pathogenic avian influenza H5N6 in Vietnam (Rounds 4–6). Figure 4 \| Province-level empirical Bayes risk of highly pathogenic avian influenza H5N6 in Vietnam (Rounds 4–6). temperatures are higher in southern than northern provinces of Vietnam. However, due to limitations in sampling strategy, with time gaps in surveillance and variation in sampling strategy between years, it is not possible to reliably assess consistency of seasonal patterns over time. production location of poultry from which samples were col- lected was not obtained during the study. Data used in the models are reflective of the locality of the market, but not necessarily the location of production. Therefore, caution is necessary regarding the interpretation of the association between risk of a market being positive for H5N1 or H5N6 and variables relating to loca- tion of production including chicken density, duck density, and the suitability of land for rice production. The prevalence of HPAI at the LBM level will be impacted by the catchment area and the extent of interconnectedness with other LBM through poultry trade. Identification of production location would enable capture of risk associated with production factors with greater accuracy. Rice-cropping intensity has previously been associated with H5N1 presence in South East Asia (18). None of the samples col- lected on the 28 market days in areas with poor suitability for rice production tested positive for H5N1. Similarly, of the six market days sampled for H5N6 in areas with poor suitability for rice production, no samples tested positive. Frontiers in Veterinary Science \| www.frontiersin.org DISCUSSION However, a significant association between the risk of H5N1-positive market days and the higher suitability of land for growing rice was not identified in this study, which may be due to the small number of market days sampled in poor rice production areas. Remote sensing data can capture greater resolution compared with traditional census collection, allowing for greater accuracy in assessment of rice-cropping intensity and suitability. This finer scale resolution may improve detection of associations between rice growing and prevalence of HPAI (18).h Reduced travel time to a major city has been associated with higher risk of H7N9 presence in LBM in Asia (34). Travel time to the nearest city is a measure of accessibility of the LBM, and the increased risk associated with more accessible LBM could be reflective of birds being drawn from more diverse populations, over a larger catchment area and connections with other LBM. In the multivariable INLA model, shorter travel time to the nearest city was not significantly associated with higher H5N1-positive market-day status. This may be due to the highly connected nature of LBM in Vietnam, enabling dissemination even between relatively less well accessed markets (12, 13).h The purpose of inclusion of variables such as chicken density, duck density, and rice suitability was to capture risk factors at point of production. Consideration must be given to the potential for chicken and duck farms to be located in geographically distant areas from the market (33). Contrary to findings of some previous studies in South East Asia, higher domestic chicken population density and waterfowl density were not found to be associated with increased risk of H5N1-positive market days (8, 17). The The residual spatial variation in H5N1 market-day risk at the province level indicates that there are unexplained fac- tors contributing to risk that were not included in the model. Vaccination has been used to control HPAI in Vietnam and may have contributed to the spatial and temporal variation in risk, April 2018 \| Volume 5 \| Article 51 8 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. Figure 5 \| Local Indicators of Spatial Association cluster maps and Moran’s I statistics of empirical Bayes risk estimates of highly pathogenic avian influenza H5N6 for Rounds 4–6. DISCUSSION Figure 5 \| Local Indicators of Spatial Association cluster maps and Moran’s I statistics of empirical Bayes risk estimates of highly pathogenic avian influenza H5N6 Figure 5 \| Local Indicators of Spatial Association cluster maps and Moran’s I statistics of empirical Bayes risk estimates of highly pathogenic avian influenza H5N6 for Rounds 4–6. Figure 6 \| Getis-Ord GI* statistic maps showing hot-spot provinces for highly pathogenic avian influenza H5N6 empirical Bayes risk estimates for Rounds 4–6. April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org H5N1 and H5N6 Epidemiology Vietnam Mellor et al. Table 4 \| Posterior mean coefficients, odds ratios (ORs), and 95% credible intervals (CrI) of spatial and non-spatial conditional autoregressive models of market days positive for highly pathogenic avian influenza H5N1 virus (Vietnam, 2011–2015). Frontiers in Veterinary Science \| www.frontiersin.org DISCUSSION Coefficient, posterior mean (95% CrI) OR, posterior mean (95% CrI) Multivariable model (no spatially varying random effect) Multivariable model (province as spatially varying random effect) Multivariable model (no spatially varying random effect) Multivariable model (province as spatially varying random effect) Suitability for rice growing High/moderate Baseline Baseline Baseline Baseline Marginal/unsuitable 0.47 (0.15, 0.78) −0.04 (−0.51, 0.42) 1.60 (1.17, 2.19) 0.96 (0.60, 1.52) Sampling month January Baseline Baseline Baseline Baseline February −0.27 (−0.67, 0.12) −0.36 (−0.77, 0.05) 0.76 (0.51, 1.13) 0.70 (0.46, 1.05) March −0.87 (−1.40, −0.36) −1.00 (−1.55, −0.47) 0.42 (0.25, 0.70) 0.37 (0.21, 0.63) April −1.01 (−1.55, −0.49) −1.11 (−1.68, −0.57) 0.37 (0.21, 0.62) 0.33 (0.19, 0.57) May −1.48 (−2.19, −0.82) −1.75 (−2.49, −1.06) 0.23 (0.11, 0.44) 0.17 (0.08, 0.35) June −0.27 (−1.19, 0.60) −0.59 (−1.51, 0.29) 0.76 (0.30, 1.83) 0.55 (0.22, 1.34) July −0.50 (−1.47, 0.43) −0.82 (−1.79, 0.12) 0.61 (0.23, 1.54) 0.44 (0.17, 1.13) August −0.35 (−1.26, 0.53) −0.65 (−1.58, 0.26) 0.70 (0.20, 1.70) 0.52 (0.21, 1.30) September −0.80 (−1.60, −0.05) −1.10 (−1.92, −0.33) 0.45 (0.20, 0.95) 0.33 (0.15, 0.72) October −1.80 (−3.44, −0.49) −1.96 (−3.62, −0.62) 0.16 (0.03, 0.61) 0.14 (0.03, 0.54) November −0.53 (−0.99, −0.08) −0.66 (−1.13, −0.20) 0.59 (0.37, 0.92) 0.52 (0.32, 0.82) December −0.80 (−1.20, −0.40) −0.92 (−1.34, −0.51) 0.45 (0.30, 0.67) 0.40 (0.26, 0.60) Average maximum temperature (°C) ≤24.47 Baseline Baseline Baseline Baseline 24.48–28.74 0.72 (0.26, 1.20) 0.15 (−0.75, 1.03) 2.06 (1.29, 3.34) 1.17 (0.47, 2.81) 28.75–30.32 1.71 (1.29, 2.15) 1.12 (0.21, 1.99) 5.54 (3.63, 8.62) 3.07 (1.23, 7.32) ≥30.33 1.69 (1.23, 2.16) 1.21 (0.26, 2.12) 5.41 (3.43, 8.71) 3.36 (1.29, 8.36) River density (km length/km2) 0.08 (−0.05, 0.21) −0.05 (−0.29, 0.19) 1.08 (0.95, 1.23) 0.95 (0.75, 1.21) Travel time (min) to nearest city with population ≥50,000 −0.18 (−0.33, −0.04) −0.07 (−0.24, 0.08) 0.84 (0.72, 0.96) 0.93 (0.78, 1.09) Chicken density (heads/km2) <285 Baseline Baseline Baseline Baseline 285–791.3 0.19 (−0.13, 0.51) 0.03 (−0.39, 0.45) 1.21 (0.88, 1.66) 1.03 (0.68, 1.57) 791.4–1,686.1 0.07 (−0.27, 0.41) 0.00 (−0.41, 0.42) 1.07 (0.77, 1.50) 1.00 (0.66, 1.52) ≥1,686.2 −0.44 (−0.86, −0.03) −0.37 (−0.87, 0.12) 0.64 (0.42, 0.97) 0.69 (0.42, 1.13) Round Round 1 Baseline Baseline Baseline Baseline Round 2 1.11 (0.76, 1.46) 1.14 (0.76, 1.52) 3.02 (2.13, 4.31) 3.13 (2.14, 4.57) Round 3 0.85 (0.48, 1.21) 0.85 (0.47, 1.24) 2.33 (1.62, 3.35) 2.35 (1.60, 3.56) Round 4 −0.12 (−0.85, 0.61) 0.06 (−0.68, 0.81) 0.89 (0.43, 1.85) 1.06 (0.51, 2.25) Round 5 0.68 (−0.02, 1.33) 0.76 (0.03, 1.44) 1.97 (0.98, 3.78) 2.14 (1.03, 4.23) Round 6 1.25 (0.18, 2.29) 1.57 (0.47, 2.65) 3.48 (1.19, 9.91) 4.81 (1.60, 14.16) Model deviance information criterion 1,934.81 1,822.10 Province included as a spatially varying random effect. DISCUSSION April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 10 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. Table 5 \| Posterior mean coefficients, odds ratios (ORs), and 95% credible intervals (CrI) of non-spatial conditional autoregressive models of market days positive for highly pathogenic avian influenza H5N6 virus (Vietnam, 2011–2015). Coefficient, posterior mean (95% CrI) Multivariable model (no spatially varying random effect) OR, posterior mean (95% CrI) Multivariable model (no spatially varying random effect) River density (km length/km2) −0.74 (−1.17, −0.34) 0.48 (0.31, 0.71) Human population density (heads/km2) 0.28 (−0.08, 0.61) 1.31 (0.92, 1.84) Market density (live bird market/10 km2) ≤2.8 Baseline Baseline 2.81–4.64 −0.15 (−1.45, 1.16) 0.86 (0.24, 3.19) 4.65–8.91 0.90 (−0.24 2.14) 2.47 (0.78, 8.52) ≥8.92 0.28 (−1.01, 1.61) 1.32 (0.36, 4.99) Round Round 4 Baseline Baseline Round 5 3.37 (2.39, 4.48) 26.06 (9.84, 78.44) Round 6 3.72 (2.62, 4.92) 40.48 (13.61, 133.16) Model deviance information criterion 200.94 Table 5 \| Posterior mean coefficients, odds ratios (ORs), and 95% credible intervals (CrI) of non-spatial conditional autoregressive models of market days positive for highly pathogenic avian influenza H5N6 virus (Vietnam, 2011–2015). Sampling at the district level was randomised for Round 1, then from Round 2 onwards the sampling strategy at the district level was to target districts with higher risk of H5N1 infection. The variation in odds of a market-day testing positive for H5N1 or H5N6 between Rounds may reflect temporal variation in risk, however, is augmented by the differences in sampling strategies implemented in different rounds. In addition, the sampling strategy at the level of the LBM was not perfectly sensitive; not all birds were sampled and AI positive birds may have been clustered in only part of an LBM. Due to the potential for under-detection, the observed proportion of HPAI positive market days may be lower than the true proportion of HPAI positive market days. In addition, aggregating market days by province, for reasons of anonymity, will have resulted in some loss of within-province het- erogeneity. However, as the provinces with the highest risk were also the smallest (southern) provinces, this loss of information is expected to be comparatively small. In conclusion, this study suggests that the spatial patterns and risk factors are very different for H5N1 and H5N6 in Vietnam. Whilst H5N1 distribution was spatially heterogeneous with sig- nificant clustering of high-risk provinces in the south, H5N6 was homogenously distributed. AUTHOR CONTRIBUTIONS PL, SN, KI, and PP contributed to the design of the study. KS, KM, AM, DE, GF, and DP contributed to the data analysis plan. AM organised the database. KS, KM, AM, and DE performed the statistical analyses. KM wrote the first draft of the manuscript. KS and AM wrote sections of the manuscript. KS, KM, AM, GF, DP, TV, and MG contributed to interpretation of results. All the authors contributed to manuscript revision, read and approved the submitted version. Figure 7 \| Choropleth map showing the province-level posterior mean probabilities of the spatially structured random effect for H5N1. DISCUSSION In addition, the likelihood of H5N1 detection at LBM was primarily associated with climatic factors. The different epidemiology of these two HPAI virus strains in Vietnam suggests the need for different surveillance and control approaches. Figure 7 \| Choropleth map showing the province-level posterior mean probabilities of the spatially structured random effect for H5N1. DISCUSSION ), and 95% credible intervals (CrI) of spatial and non-spatial conditional autoregressive models of market days (Vietnam, 2011–2015). Table 4 \| Posterior mean coefficients, odds ratios (ORs), and 95% credible intervals (CrI) of spatial and non-spatial conditional autoregressive models of market days positive for highly pathogenic avian influenza H5N1 virus (Vietnam, 2011–2015). Table 4 \| Posterior mean coefficients, odds ratios (ORs), and 95% credible intervals (CrI) of spatial and non-spatial conditional autoregressive models of market days positive for highly pathogenic avian influenza H5N1 virus (Vietnam, 2011–2015). Province included as a spatially varying random effect. Previous studies mapping the spatial distribution of avian influenza in Vietnam have utilised data obtained through passive surveillance (8, 18). In Vietnam, passive surveillance is conducted through farmers or community animal health workers notifying local state vets, with subsequent diagnostic testing and investiga- tion. Positive samples are then reported to the central govern- ment. Currently, a low number of outbreaks are reported through passive surveillance (32) due to the reliance on clinical detection of disease, testing, and reporting processes. During this study, samples were collected through active surveillance, with stand- ardised selection criteria across regional areas within each round of sample collection. This approach enables detection of HPAI in subclinical birds and minimises temporal and spatial variation in surveillance effectiveness, enabling more robust identification of regional differences in the prevalence of H5N1 and H5N6 at the level of the LBM. The ongoing active surveillance conducted as vaccine coverage has been found to vary with both district and season (35). In addition, the predominant duck breeds may vary between regions and vaccine response of different breeds of domestic ducks to the commercial vaccines has been shown to differ, resulting in shedding continuing in some clinically unaf- fected, vaccinated ducks (36–38). Additional market level factors not included in the model have the potential to contribute to between-market variation in the likelihood of a sample testing positive for H5N1 or H5N6. Factors include the number of days per week the market opens, biosecurity measures, length of holding of birds in the LBM, number of birds and stocking density in the LBM, biosecurity, and husbandry on farms producing the poultry for sale (39, 40). Collection of further market level information would enable further improvement of understanding of both H5N1 and H5N6 epidemiology in Vietnam. ETHICS STATEMENT Data were generated as part of routine governmental active sur- veillance monitoring of avian influenza in Vietnam, coordinated by the Department of Animal Health, Ministry of Agriculture and Rural Development in Vietnam. All surveillance activi ties and protocols were approved by the Vietnamese Depart ment of Animal Health (DAH) Epidemiology Division before implementation. 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This work was partially supported by USAID (grant number: GHA-G-00-06-00001). The authors would like to thank USAID for their generous support, which contributed significantly to the success of this operational research and has helped to build in LBM is essential to monitor changes in spatiotemporal distri- bution patterns and strain evolution. Sampling continues to be focussed upon LBM and provinces where prevalence of infection has previously been detected to be highest. One of the main limitations of the data analysed in this study is that the sampling strategy was not consistent between Rounds. April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 11 H5N1 and H5N6 Epidemiology Vietnam Mellor et al. Council (MRC), and Natural Environment Research Council (NERC): http://www.bbsrc.ac.uk/research/international/zels/. MG is funded by the Belgian FNRS, and this work was partly sup- ported by the NIH grant 1R01AI101028-02A1. 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Transbound Emerg Dis (2016) 63:127–35. doi:10.1111/tbed.12470 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 36. Cagle C, To T, Nguyen T, Wasilenko J, Adams S, Cardona C, et al. Pekin and Muscovy ducks respond differently to vaccination with a H5N1 highly pathogenic avian influenza (HPAI) commercial inactivated vaccine. Vaccine (2011) 29:6549–57. doi:10.1016/j.vaccine.2011.07.004 Copyright © 2018 Mellor, Meyer, Elkholly, Fournié, Long, Inui, Padungtod, Gilbert, Newman, Vergne, Pfeiffer and Stevens. 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 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 © 2018 Mellor, Meyer, Elkholly, Fournié, Long, Inui, Padungtod, Gilbert, Newman, Vergne, Pfeiffer and Stevens. 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 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. 37. Steensels M, Van Borm S, Lambrecht B, De Vriese J, Le Gros F, Bublot M, et al. REFERENCES Interventions for avian influenza A (H5N1) risk management in live bird market networks. Proc Natl Acad Sci U S A (2013) 110:9177–82. doi:10.1073/ pnas.1220815110 27. Hijmans R, Cameron S, Parra J, Jones P, Jarvis A. Very high resolution interpo- lated climate surfaces for global land areas. Int J Climatol (2005) 25:1965–78. doi:10.1002/joc.1276 28. ESRI. ArcGIS Desktop: Release 10.3.1. Redlands, CA: Environmental Systems Research Institute (2015). 13. Wang J, Vijaykrishna D, Duan L, Bahl J, Zhang J, Webster R, et al. Identification of the progenitors of Indonesian and Vietnamese avian influenza A (H5N1) viruses from Southern China. J Virol (2008) 82:3405–14. doi:10.1128/ jvi.02468-07 29. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing (2017). Available from: https://www.r-project.org/ (Accessed: June 30, 2017). j 14. Leung Y, Lau E, Zhang L, Guan Y, Cowling B, Peiris J. Avian influenza and ban on overnight poultry storage in live poultry markets, Hong Kong. Emerg Infect Dis (2012) 18:1339–41. doi:10.3201/eid1808.111879 30. Calcagno V, Mazancourt C. glmulti: an R package for easy automated model selec- tion with (generalized) linear models. J Stat Softw (2010) 34:1–29. doi:10.18637/ jss.v034.i12 15. Nguyen D, Uyeki T, Jadhao S, Maines T, Shaw M, Matsuoka Y, et al. Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001. J Virol (2005) 79:4201–12. doi:10.1128/jvi.79.7.4201-4212.2005 31. Rue H, Martino S, Chopin N. Approximate Bayesian inference for latent Gaussian models by using integrated nested Laplace approximations. J R Stat Soc Series B Stat Methodol (2009) 71:319–92. doi:10.1111/j.1467-9868.2008. 00700.x April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 12 Mellor et al. H5N1 and H5N6 Epidemiology Vietnam Asian H5N1 HPAI. Vaccine (2009) 27:646–54. doi:10.1016/j.vaccine.2008. 11.044 32. Delabouglise A, Choisy M, Phan T, Antoine-Moussiaux N, Peyre M, Vu T, et al. Economic factors influencing zoonotic disease dynamics: demand for poultry meat and seasonal transmission of avian influenza in Vietnam. Sci Rep (2017) 7:5905. doi:10.1038/s41598-017-06244-6 39. Kung N, Guan Y, Perkins N, Bissett L, Ellis T, Sims L, et al. The impact of a monthly rest day on avian influenza virus isolation rates in retail live poultry markets in Hong Kong. Avian Dis (2003) 47:1037–41. doi:10.1637/0005-2086- 47.s3.1037 33. Fournié G, Tripodi A, Nguyen T, Nguyen V, Tran T, Bisson A, et al. Frontiers in Veterinary Science \| www.frontiersin.org REFERENCES Efficacy of an inactivated and a Fowlpox-vectored Vaccine in Muscovy Ducks against an Asian H5N1 highly pathogenic avian influenza viral challenge. Avian Dis (2007) 51:325–31. doi:10.1637/7628-042806r.1 38. Steensels M, Bublot M, Van Borm S, De Vriese J, Lambrecht B, Richard-Mazet A, et al. Prime–boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with 38. Steensels M, Bublot M, Van Borm S, De Vriese J, Lambrecht B, Richard-Mazet A, et al. Prime–boost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with April 2018 \| Volume 5 \| Article 51 Frontiers in Veterinary Science \| www.frontiersin.org 13
W4237304460.txt	https://www.nomos-elibrary.de/10.5771/0023-4834-1999-1-132.pdf	de		Die Kritik der Kritik der Kritik - Ein Glückwunschbeitrag zur Dreißigjahrfeier der Kritischen Justiz -	Kritische Justiz	1,999	cc-by	2,850	IJ2 geographische Distanzen hinweg. Darin liegt die große zivilisatorische Leistung des Rechts, die bereits das Römische Reich ermöglicht und zusammengehalten hat. Nun stellen wir fest, daß sowohl innerhalb der modernen Gesellschaften wie auch in den internationalen Beziehungen Fremdheit immer stärker nachgelassen hat. Durch die weltumspannenden Kommunikationsmedien, durch Tourismus, zunehmend auch durch das Internet werden Individuen, Gruppen, Firmen und Organisationen derart in Verbindung miteinander gebracht, daß durchaus so etwas wie Vertrautheit miteinander entsteht. Es ist natürlich keine persönliche Vertrautheit, sondern eine funktionale, die sich auf den Charakter der jeweiligen Beziehung erstreckt, aber sie reicht i. d. R. aus, um verläßliche Interaktionen zu ermöglichen. Jedenfalls hat es den Anschein, als ob das Recht als das verbindende Element sozial und geographisch entfernter Partner an Bedeutung verliert. Nicht zufällig gibt es unter vielen Nutzern des Internet weltweit eine breite Stimmung gegen jegliche Art von rechtlicher Regulierung der Nutzung dieses neuen weltumspannenden Mediums. Sie meinen, daß das Medium selbst gewissermaßen die grundlegenden sozialen Voraussetzungen für seine verantwortliche Nutzung bereits enthält. Das mag richtig sein oder nicht, jedenfalls drückt diese Auffassung eine Tendenz aus, Vertrauen unter Kommunikationspartnern auf andere Weise als durch die Erwartungssicherheit des Rechts zu erzeugen. Wo die Prozesse der Globalisierung prinzipiell alle Menschen einander nahegebracht und jedenfalls Fremdheit aus geographischen Gründen weitgehend beseitigt haben, da verliert das Medium, das bislang soziale Kommunikation und Kooperation über große Distanzen ermöglicht hat, an Bedeutung. Doch mag das auch mit einer antizivilisatorischen Tendenz einhergehen, wonach sozialer Austausch die Überwindung von Fremdheit, nicht deren Anerkennung, verlange. Es könnte dazu kommen, daß nur einander Vertraute verläßliche soziale Beziehungen miteinander eingehen, und diese Vertrautheit kann auf gemeinsame Kulturen, Religion, Abstammung oder andere askriptive Eigenschaften gegründet sein. Das positive Recht hat mit seinen Abstraktionen historisch derartige Verengungen überwunden. Vielleicht werden wir demnächst einmal ein Medium brauchen, das Fremdheit und Distanz wieder herstellt, die Qualität also, die in der Vergangenheit mit dem Begriff der Zivilität umschrieben worden ist. Hierin - in der Bewährung dieser großen Leistung der Vergangenheit könnte, trotz aller gegenläufigen Tendenzen, die Zukunft des Rechts liegen. Ivana MikeSic Die Kritik der Kritik der Kritik - Ein Glückwunschbeitrag zur Dreißigjahrfeier der Kritischen Justiz »Welchen sollen wir toten?« Bertolt Brecht, Dreigroschenoper I. Szenario Frankfurt, ein Nachmittag im Oktober 1998. Die Kritische Justiz feiert Geburtstag. Und alle, alle sind sie gekommen. Auftakt: Festansprache des Gastgebers und Gründungsmitgliedes der Zeitschrift, Festansprache des Verlegers, Grußwort des Justiz- https://doi.org/10.5771/0023-4834-1999-1-132 Generiert durch IP '51.159.168.146', am 14.07.2024, 04:10:22. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. ministers . Dann der Dialog der Generationen. In guter Frankfurter Manier wird nicht mit dem kunstvoll gedrechselten Festvortrag von Professor X gefeiert, sondern mit einer großen Podiumsdiskussion. Im Plenum di e Gründergeneration der Zeitschrift, Redakteure wie Autoren: Professoren, Rechtsanwälte, Richter, darunter zwei des Bundesverfassungsgerichts, und nicht zu vergessen der schon erwähnte Minister; nur wenige Assistenten., einige Studenten. Frauen in erklecklicher Zahl, immer noch nicht genug. Auf dem Podium ein Querschnitt durch den Mittelbau der Universität Frankfurt, junge ReferendarInnen und Assistentlnnen. I Ihre Aufgabe ist es, recht kritisch zu sein zu Ehren der Kritischen Justiz (respektive ihrer im Plenum weilenden Mütter und Väter). Ein Papier wird verlesen, es wird angemessen radikaldemokratisch und mit unverkennbarer Streitlust dargeboten. Dann eine lebhafte, im besten Sinne festliche Diskussion: Die kritischen Juristen der ersten Stunde kritisieren die Kritik ihrer in der Kritik noch nicht ganz so souverän bewanderten akademischen Sprößlinge, die Sprößlinge halten, so gut es eben geht, dagegen. Am Ende ist alles sehr schön und so, wie es sein soll. Das Podium fühlt sich ein bißchen mißverstanden, aber doch geehrt durch di e Aufmerksamkeit der illustren Festgäste, das Plenum hat es den jungen Aufstrebenden nochmal richtig gezeigt - und die Autorin erinnert sich nach längerer Abwesenheit aus den Frankfurter Diskussionszusammenhängen und in einem seltsamen Anfall von Wehmut wieder daran, warum sie die Heroen ihrer juristischen Kleinkindzeit toll fand und immer noch findet. 2. Erinnerung Das juristische Gedächtnis der Autorin reicht nicht wie das der Jubilarin dreißig Jahre zurück, sondern in etwa zehn: Frankfurt, im Sommersemester 1988. Parallel zum zuvor bereits angefangenen Philosophiestudium machte man sich - mit nicht wenigen gleichgesinnten rechtstheoretisch Begeisterten - auf, das Kommunikationsparadigma am und im Jurastudium zu erproben. Frankfurt lockte. Eine philosophische Fakultät, die auch Ende der achtziger Jahre noch von der Anziehungskraft der Kritischen Theorie profitierte (auch wenn man das daselbst nicht immer wahrhaben wollte). Zumal eine Fakultät, der Jürgen Habermas einen der bislang letzten großen, interdisziplinär wirksamen Theorieschübe verpaßte, bevor man in den einzelnen Geistesoder Kulturwissenschaften wieder begann, sich vom zeitweiligen, teilweise euphorisierten theoretischen Primat der Sozialwissenschaften und der Philosophie auf ureigene Forschungsgegenstände zurückzuziehen. Zum anderen, und in wunderbarer Ergänzung, die juristische Fakultät: Der Ruf der Liberalität, des soziologisch, historisch und philosophisch sensibilisierten Umgangs mit d er Wissenschaft vom Recht übte seine Wirkung auf gymnasiale Hitzköpfe ebenso wie auf junge ambitionierte Wissenschaftler. Interdisziplinäre Offenheit war Ehrensache. Seinen Niederschlag in der Studienordnung fand dieser Ansatz in einem ersten, auf die Grundlagenfächer (Sozialwissenschaften, Methodologie, Geschichte) konzentrierten Orientierungssemester, didaktischer Kern der Frankfurter juristischen Ausbildung. Persönliche Gewähr für dessen Attraktivität leistete ein Kreis von Hochschullehrern., die es vermochten, zumindest noch in Momenten den Begeisterungsfähigen unter ihren Studentinnen und Studenten den Eindruck einer gemeinsamen Programmatik zu vermitteln - es war eine letzte Blüte: Wie die geneigte Leserschaft weiß, ist das Grundlagenmodell mittlerweile Geschichte. Als zentrales Publikationsorgan jener I Welch eigentumlichen Reiz diese Konstellation trotz - oder wegen - ihrer so offensichtlichen Inszeniertheit übte, laßt sich auch aus der Beschreibung von Uwe Wesel, Wenn die Linken Schlipse tragen, DIE ZEIT Nr. 49 vom 26. November 1998, S. 51 erspuren. H err Wesel trug ubrigens keinen Schlips, aber er kam im Anzug! https://doi.org/10.5771/0023-4834-1999-1-132 Generiert durch IP '51.159.168.146', am 14.07.2024, 04:10:22. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 133 134 theoretisch aufgeschlossenen Juristen (natürlich auch über Frankfurt hinaus) galt die Kritische Justiz, und deren Lektüre wurde eindringlich und selbstbewußt empfohlen. Für die Autorin steht außer Frage: Sie möchte zu keiner anderen Zeit an keinem anderen Ort studiert haben. Freilich: Mancher der Lehrenden vergaß in seiner Begeisterung, über aller Kritik den Studierenden das Subsumieren beizubringen. So mag in dem einen oder anderen Fall die Lossagung von jeglicher spezifisch juristischer Wissensvermittlung entlastend auf die persönliche Terminplanung gewirkt haben. U nvergeßlich die schöne Vorlesung, in der ein Hochschullehrer den möglichst frühzeitigen Besuch beim Repetitor empfahl, der doch so hervorragende und professionelle Ausbildungsarbeit leiste, während man sich an der Universität mit den wirklich wichtigen Dingen des Lebens beschäftigen könne. Unnachahmlich auch die Nonchalance, mit der sich einer seiner Kollegen die künftige Anwesenheit einiger zur ersten Vorlesungsstunde im Semester erschienener Ahnungsloser verbat mit der Begründung, der größte Teil von ihnen sei intellektuell ohnehin nicht in der Lage, seinen Ausführungen zu folgen. Von den sonstigen strukturellen und personellen Unzulänglichkeiten der damals endgültig ausbrechenden Massenuniversität wollen wir hier schweigen - darüber reden wir schließlich jeden Tag, und dies ist ein Festbeitrag. Es gab neben und trotz alledem diejenigen, die die Kärrnerarbeit auf sich nahmen: Wissensvermittlung von der Pike auf, immer wieder von vorne, gründlich, systematisch, kritisch und zur Kritik motivierend, im Vertrauen auf die geistige, moralische und politische Entwicklungsfähigkeit der Studierenden. Die, denen ein kluges und gut plaziertes Argument mehr zählte als ein Haufen auswendig gelernter Definitionen. Die Momente der kritischen Distanz auch zu sich und ihrem Fach aufscheinen lassen konnten. Die haben es gelohnt. Das war sie, Ende der achtziger Jahre, die Gründergeneration der Kritischen Justiz, die, wie Wes el sie nennt, »juristischen Rebellen« von 1968: und zwar in ihrer ersten Phase der institutionellen Etablierung: an den Universitäten. Was ist heute? J. Kritik Der inszenierte G eburtstags-Generationenkonflikt hatte seine ernsthaften Wendungen, auch wenn es im wesentlichen bei einer leidlich harmlosen Tändelei blieb. Die kämpferischen Momente hatten überwiegend spielerischen Charakter, hier machte sich die allgemeine Festtagslaune bemerkbar. Alles in allem muß man wohl sagen, daß das Plenum Sieger nach Punkten wurde, während das Podium in der Argumentation (also im Nahkampf) das aggressive Potential seines Thesenpapiers nicht überzeugend zu entfalten wußte. Einige Beobachtungen seien hier wiedergegeben. a) Die Kinder Bei aller Sympathie für manche Position, die das Thesenpapier bezieht (dazu gehört das Festhalten am Postulat kritisch-rechtswissenschaftlicher Analyse; das Bewußtsein für die Bedeutung informeller Diskurse; die Bereitschaft zur kontinuierlichen Reflexion der Möglichkeiten und Defizite demokratischer Durchdringung in Justiz, Wissenschaft und Politik): Es scheint darin eine Tendenz auf, die naiv und anmaßend zugleich ist. Nimmt man Autoren und Autorinnen beim Wort, wird Kritik in einer 2 Uwe Wesel (Fn. I). https://doi.org/10.5771/0023-4834-1999-1-132 Generiert durch IP '51.159.168.146', am 14.07.2024, 04:10:22. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. Absetzungsbewegung erst möglich, einzig außerhalb der Justiz, der Verwaltung, der Parlamente, Parteien und Verbände. Kann das ernst gemeint sein? Ist es Bedingung der Möglichkeit einer kritischen Position, »außen« zu sein? Darf ich mich kritisch erst nennen, wenn ich vorher den Wegzoll bezahlt habe, nämlich vorsichtshalber die Institution, für die ich arbeite, als per se undemokratisch gebrandmarkt habe? Wozu sonst soll das führen als zu bedauerlich nivellierendem Lagerdenken. Der strategische Fehler dieser Argumentation ist, daß die Autoren sich vom Dilemma der Kritik der Kritik haben einfangen lassen. Wie gelingt es, kritischer zu sein als die Kritischen? Man versucht erstens, die Alten mit deren eigenen Waffen zu schlagen. Problem: Die können das leider immer noch am besten (und sei es nur, den Habitus gekonnt einzunehmen). Man besetzt, zweitens, die wichtigen und ausreichend unangenehmen Themen. Problem: Dafür erreicht man nur schwer die nötige Aufmerksamkeit, oder man ergeht sich, drittens, in dem, was man für gesteigert radikal hält. Problem: Rein quantitative Methode, die selten jemand ernst nimmt. Mit allen drei Strategien scheitert das Papier. Es reproduziert einen langweiligen und abstrakten Abgrenzungsmechanismus, der der Realität des beruflichen Alltags (auch in der Wissenschaft, und, wenn die Autoren ehrlich sind, auch in ihrem eigenen Umfeld) nicht gerecht werden kann. Darüber verlieren die angeschnittenen, durchaus aktuellen Einzelprobleme (Ausbildung und Prüfung, zunehmende Steuerungsanforderungen an das Recht durch Prozesse gesellschaftlicher Umstrukturierung, beschleunigter Regulierungskreislauf Ökonomie/soziale Sicherung) an Schärfe. Die Themen, die dem Plenum wirklich wehtun, wurden offenbar verfehlt - zu wenig gereizt reagierten sie, zu lässig ihre Abwehr. Zu wenig geht es um das, was derzeit wahrscheinlich noch nicht einmal wirklich bewußt - interessiert. Dazu später. b) Die Gründer Andererseits die Gründergeneration im Plenum. Was bewegt sie? Jedenfalls nicht das, was an diesem Nachmittag geschieht. Zum Dialog oder gar Kampf fühlen sie sich nicht ernsthaft aufgerufen, der Fehdehandschuh bleibt liegen. Verändern gesteigerte Anforderungen der beruflichen Realität den Sinn für Kritik, für selbst geübte wie auch für die Kritik anderer? Die Autorin im Plenum nimmt um sich herum halb geflüsterte, leicht mokante Reaktionen wahr, in etwa mit der Tendenz »zu nebulös, zu abgelöst von der Praxis«. Es ist spürbar, wenn auch schwer zu fassen: Persönliche und kollektive Fragestellungen haben sich verändert, und es ist schmerzlich, daß dies unausgesprochen bleibt. Das Engagement der Jungen auf dem Podium wird dadurch disqualifiziert. In manchem Statement aus dem Plenum klingt ein altbekanntes Phänomen an: Hat man nicht schon längst, früher mal, klar gemacht, was kritisch sei? Können sich die Kinder, bitte schön, jetzt nicht daran halten? Wieso finden diese Kritikkinder nicht provokant, was man selbst vor dreißig Jahren hyperrevolutionär fand? Dazu eine weitere persönliche Erinnerung: Die Autorin denkt immer wieder zurück an ein Gespräch über strafrechtlichen Abolitionismus in den siebziger Jahren, das sie noch als studentische Hilfskraft mit ihrem großen Boss führen durfte; dem war es unverständlich, daß die junge Studentin der späten Achtziger die Thesen von Arno Plack allenfalls belustigend fand. Und das, obwohl er selbst sich damit längst nicht mehr auseinandersetzte. Bis heute ist zweifelhaft geblieben, ob die Elevin deshalb für doof oder für eigensinnig gelten mußte. Die Feier in Frankfurt zeigt es wieder: Man denkt eben selbst nicht ohne väterliche Rührung an die eigene rebellische Phase zurück. Wenn die Kinder kritisch sein wollen, sollen sie es uns doch erst mal nachmachen. Kritik ist ein Heimspiel. https://doi.org/10.5771/0023-4834-1999-1-132 Generiert durch IP '51.159.168.146', am 14.07.2024, 04:10:22. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 135 c) Lernerfolg: Auch ein kritischer Jurist ist ein Jurist. Auch eine kritische Juristin ist eine Juristin. Auch kritische Juristen bleiben von Generationenkonflikten nicht verschont. Bei Generationenkonflikten unter kritischen Juristen geht es genauso kompliziert zu wie anderswo. Kritik taugt vorzüglich als Label. Kritik kann ein schönes Festgewand tragen. Kritik kann Differenzierungsgewinne bewirken. Kritik kann Differenzierungsverluste bewirken. Kritik kann ausgrenzen. Kritik kann sich selbst zum Bekenntniszwang engführen. Kritik kann großartig sein. Kritische Juristen definieren gerne selbst, was kritisch ist. Auch Kritik ist deshalb ein Machtspiel: Ein Spiel um Definitionsmacht. 4. Macht Nicht wenige aus der Griindergeneration der kritischen Rechtswissenschaft und -praxis sind heute aufgestiegen zu Justizministern, Bundesverfassungsrichtern und, soweit sie es nicht damals schon waren, zu anerkannten Rechtslehrern und lehrerinnen. Nennen wir es die zweite Phase der institutionellen Etablierung. Es ist der für herausragende Juristen und Juristinnen durchaus übliche biographische Schritt hin zum Zentrum der Macht/der Mächte: Sie werden nach einer gewissen Zeit der professionellen Reife zu Politikern und Politikberatern, begehrten Gutachtern oder Feuilletonisten, hohen und höchsten Richtern, Seniorpartnern der großen Sozietäten und Direktoren der renommierten Forschungsinstitute. Es steht außer Frage: Die Kritischen partizipieren jetzt an der Macht.) Das große, unausgesprochene Rätsel dieser Geburtstagsfeier: Niemand redet darüber. Als habe der Gedanke daran in diesem Kreis unbewußt das Odium des Unanständigen: Gehen Kritik und Macht überhaupt zusammen, ist Kritik das Andere der Macht? Es gibt Möglichkeiten, dieses Thema philosophisch zu behandeln. Das interessiert hier nicht. Es sei nur beobachtet und festgehalten, wer wann mit wem worüber zu sprechen bereit ist. Keiner stellt an diesem Nachmittag explizit die Frage, wie mit Macht - mit Erwerb, Erhaltung, Verlust - umgegangen wird. Warum nicht? Wir wollen Bescheid wissen, wir wollen teilhaben, wir wollen es diskutieren - wir wollen es kritisieren! Sind die Mächtigen unter den Kritischen an diesem Nachmittag nur in ihrer Eigenschaft als Kritische da, möglicherweise in wehmütiger Erinnerung an ein abgelegtes biographisches Muster? Ist es eine Haltung, die man jetzt für bestimmte festliche Anlässe anlegt, während man in seinem zweifellos aufreibenden und konfliktreichen (Macht-)Alltag eine andere Existenz führt? Haben wir langfristig schizophrene Krankheitsverläufe zu befürchten? Fehlt es den einen an der Kraft, es zu reflektieren, ihre Konflikte offenzulegen, mit dem Faktum ihrer Macht offensiv umzugehen? Oder fehlt es den anderen an der Chuzpe, Rechenschaft darüber einzufordern? Viele Fragen, nur eine Sicherheit: Weder mit der pauschalen Forderung nach mehr Demokratie noch mit einer distanzierten Verweigerungshaltung ist diese Diskussion zu haben. Aber wir sollten sie führen. 3 Um es mit Wesel zu sagen: Sie tragen jetzt Schlips. https://doi.org/10.5771/0023-4834-1999-1-132 Generiert durch IP '51.159.168.146', am 14.07.2024, 04:10:22. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 5. Akademische Morde oder: Der Mythos fordert sein Recht Und dann werden sie mich sagen horen: »Alle.« Und wenn dann der Kopf fallt, sage ich: »Hoppla.« Bertolt Brecht, Dreigroschenoper Da war noch was. Im griechischen Mythos ist es mit dem Namen Ödipus verbunden, und die Psychoanalyse hat uns mit dem dazugehörigen Komplex bekannt gemacht. Es gehört mittlerweile zum vulgär-psychologischen Gemeingut der Szene, im Rekurs darauf die Vorstellung von der ödipalen Konstellation auf akademische Lehrer-Schüler-Verhältnisse zu übertragen. 4 Der junge Wissenschaftler muß den (Zieh- und Über-)Vater erschlagen, um seine eigene geistige und psychische Individuation zu vollenden. Das akademische Erwachsenwerden, die Ablösung von der Leitfigur geschieht durch eine (Ersatz-)Tötungshandlung. 5 Von Ferne erinnert dieser zwingend als gewaltsam zu denkende Akt - eine der wenigen breit konsentierten Möglichkeiten, in unserer Kultur das Tötungstabu sprachlich zu durchbrechen - an einen anderen Privilegierungstatbestand, nämlich den der Revolution. Wesel scheint sich, wenn er über alte und neue Juristengenerationen spricht, der Metaphorik des Umsturzes näher zu fühlen. 6 Zu Recht, schließlich geht es nicht nur um den Kampf der Generationen, sondern auch um die Frage, wer wann die besseren Zeiten einläuten darf. Ratlosigkeit auf seiten der Autorin: Sie will erwachsen werden. Muß sie jetzt töten, und wenn ja, um Himmels willen, wen? Ungern will sie sich die brüskierte Kleinmädchenphantasie der Seeräuberjenny zum Vorbild nehmen.? Vielleicht ist sie noch nicht kritisch genug, mag sein, Klassenziel nicht erreicht, das Eisen weiter schmieden. Oder zu anhänglich, in alter Verehrung für die Überpapas und Übermamas. 8 Oder winkt ihr schon verführerisch die Macht, ein großes Versprechen - möglichst schnell möglichst nah dran, vergiß, was du gelernt hast, studiere die Techniken des Macchiavell. Potzblitz, schon in zweierlei Hinsicht müssen die Altvorderen nunmehr überboten werden: Man sei kritischer als sie und werde mächtiger als sie. Vielleicht so: Sie haben uns gezeigt, wie man kritisiert. Das war schön, aber es reicht uns noch nicht. Jetzt sollen sie uns vormachen, wie man in diesem Geiste regiert und dirigiert, teilt, herrscht, mächtig ist. Wenn sie das nicht können, sollen sie es uns überlassen. 4 Diese Qualifizierung ist nicht per se negativ. Sie wird es da, wo sich »Ordinarien« als letzte Horte der Erbuntertanigkeit erweisen. 5 Es müßte sich ein re ger Geist finden, der sich die Muhe macht, auch noch alle mo glichen Geschlechterkonstellationen dieser Problematik durchzuspielen. 6 Wesel (Fn. I): Abbildungen von ehe Guevara; »Revolte«, »Rebellion«, »langer Marsch«. 7 Die als Madchen ror alles im lumpigen Hotel »das Bett fur jeden« machen muß, um spater in einer grandiosen Rache- und kaum verhullten Kastrationsphantasie ... nein, nur die Kopfe rollen zu lassen. 8 S.o., I. und 3. https://doi.org/10.5771/0023-4834-1999-1-132 Generiert durch IP '51.159.168.146', am 14.07.2024, 04:10:22. Das Erstellen und Weitergeben von Kopien dieses PDFs ist nicht zulässig. 137
https://openalex.org/W2416125190	https://europepmc.org/articles/pmc5089579?pdf=render	English	null	Directional deep brain stimulation of the subthalamic nucleus: A pilot study using a novel neurostimulation device	Movement disorders	2,016	cc-by	3,662	Directional Deep Brain Stimulation of the Subthalamic Nucleus: A Pilot Study Using a Novel Neurostimulation Device DBS of the STN has proven to be a safe, effective treatment for patients with severe tremor, motor ﬂuctuations, or dyskinesia in Parkinson’s disease.1,2 However, individual outcomes may vary greatly and critically depend on the brain volume being stimulat- ed. Best motor symptom control has been associated with stimulation of the dorsolateral STN,3,4 whereas current leaking into adjacent ﬁber tracts can cause adverse effects such as dysarthria, impaired ﬁne motor control, or oculomotor disturbances.5 Frank Steigerwald, MD,1 Lorenz M€uller, MD,1 Silvia Johannes, MD,2 Cordula Matthies, MD, PhD,2 and Jens Volkmann, MD, PhD1* Conventional DBS systems use ring-shaped electrodes, which generate an approximately spherical electrical ﬁeld. In these systems, programming of polarity and stimulation pulse parameters allows only limited control of the shape of the volume of tissue activated.6 Recently, two acute intraoperative studies have proven the feasi- bility of horizontal current steering by using novel lead designs, such as segmented or multicontact electrodes.7,8 Directed stimulation using these electrodes resulted in increased stimulation thresholds for side effects as com- pared to standard spherical stimulation. 1Department of Neurology, University Clinic of W€urzburg, W€urzburg, Germany 2Department of Neurosurgery, University Clinic of W€urzburg, W€urzburg, Germany Supporting Data window in a monopolar review as compared to ring- mode DBS. V C 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of Inter- national Parkinson and Movement Disorder Society Key Words: deep brain stimulation; Parkinson’s disease window in a monopolar review as compared to ring- mode DBS. V C 2016 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of Inter- national Parkinson and Movement Disorder Society Key Words: deep brain stimulation; Parkinson’s disease Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. S T E I G E R W A L D E T A L S T E I G E R W A L D E T A L ABSTRACT Introduction: A novel neurostimulation system allows steering current in horizontal directions by combining segmented leads and multiple independent current control. The aim of this study was to evaluate direc- tional DBS effects on parkinsonian motor features and adverse effects of subthalamic neurostimulation. Here, we report our ﬁrst clinical experience of direc- tional DBS with a novel, fully implantable neurostimu- lation system (Vercise PC; Boston Scientiﬁc, Valencia, CA), which combines eight-contact directional leads and a pulse generator capable of multiple independent current source control (MICC). The system received a CE Mark in September 2015, and the ﬁrst device was implanted at our center on 16 September 2015. Methods: Seven PD patients implanted with the novel directional DBS system for bilateral subthalamic DBS underwent an extended monopolar review session dur- ing the ﬁrst postoperative week, in which current thresholds were determined for rigidity control and stimulation-induced adverse effects using either direc- tional or ring-mode settings. p p The novel directional DBS lead has four electrode levels, of which the two middle levels are split into three segments spanning approximately 120 degrees, whereas the highest and lowest level consist of ring- shaped electrodes (Supporting Fig. 1A). MICC allows to distribute the stimulation current over any combi- nation of electrodes of one lead in arbitrary propor- tions. An equal distribution of current among all three segments at one level simulates a ring-shaped elec- trode, whereas maximal horizontal steering effects are obtained when current is distributed to one or two segments at one level (Supporting Fig. 1B). Our retro- spective analysis of monopolar review data aimed at quantifying the effect of horizontal current steering on the therapeutic window of STN-DBS as compared to conventional ring mode stimulation. Results: Effect or adverse effect thresholds were modi- ﬁed by directional settings for each of the 14 STN leads. Magnitude of change varied markedly between leads, as did orientation of optimal horizontal current steering. Published online 31 May 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.26669 ABSTRACT Conclusion: Directional current steering through chron- ically implanted segmented electrodes is feasible, alters adverse effect and efﬁcacy thresholds in a highly individual manner, and expands the therapeutic ------------------------------------------------------------ This is an open access article under the terms of the Creative Commons Conclusion: Directional current steering through chron- ically implanted segmented electrodes is feasible, alters adverse effect and efﬁcacy thresholds in a highly individual manner, and expands the therapeutic ------------------------------------------------------------ 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. Correspondence to: Prof. Dr. Jens Volkmann, Department of Neurol- ogy, University Clinic of W€urzburg, Josef Schneider Strasse 11, D-97080 W€urzburg, Germany; E-mail: volkmann_j@ukw.de Correspondence to: Prof. Dr. Jens Volkmann, Department of Neurol- ogy, University Clinic of W€urzburg, Josef Schneider Strasse 11, D-97080 W€urzburg, Germany; E-mail: volkmann_j@ukw.de Relevant conﬂicts of interest/ﬁnancial disclosures: Nothing to report. Full ﬁnancial disclosures and author roles may be found in the online ver- sion of this article. Results We could determine efﬁcacy thresholds for only 11 of 14 STNs, because a persistent microlesioning effect pre- vented reliable rigidity assessment in the others, whereas AE thresholds were determined for all 14 STNs. The AEs determining the upper limit of the TW were contractions of facial or hand muscles in 11 of 14 STNs, dysarthria in 6 of 14, and persisting dysesthesia in 1 of 14. The programming session followed the procedure of a standard monopolar review,6 in which for each electrode conﬁguration current thresholds are determined for com- plete rigidity control (efﬁcacy threshold) and the ﬁrst adverse event (AE) limiting further current increase (AE threshold). Frequency and pulse width were constantly set to 130 Hz and 60 ls, respectively, and the implant- able pulse generator case was always programmed as anode. Stimulation current was increased/decreased in steps of 0.5 mA until complete rigidity suppression (i.e., hypotonia of the upper extremity) was achieved or an AE of stimulation was reported by the patient or observed on clinical examination. Then, the current threshold was ﬁne tuned in smaller steps of 0.1 mA. , p g y In total, we assessed 154 directional and 28 ring- mode settings and determined TW for 111 and 24 set- tings, respectively. For each directional setting, the proportional change of TW from corresponding ring- mode stimulation was calculated, with negative values indicating a reduction and positive values indicating an increase of the TW. The proportional change was highly variable between leads and directional settings and ranged between –100% and 440% (see Table 1). Interestingly, this change in TW was not only deter- mined by a variable AE threshold, but also by varia- tions in the effect threshold (see Supporting Fig. 3). Using this procedure, we ﬁrst determined the thera- peutic window (TW; current difference between efﬁca- cy and AE threshold in mA) for the two segmented levels of each lead in ring mode (equally distributing current among the three segments of a level) and labeled the level with the larger TW as “most effective ring level.” Then, different stimulation directions at each level were tested by either restricting cathodal current to each of the three segments (ﬁrst patient) or additionally evaluating equal current distributions between two adjacent segments (subsequent 6 patients). This programming results in up to six direct- ed electrical ﬁelds with a ﬁeld vector rotated by either 120 or 60 degrees (Supporting Figs. Patients and Methods Best and worst effect on TW Changes in therapeutic window (DTW) in best and worst direction and respective orientation of electrical ﬁeld vector are given for all STN in which effect and AE threshold could be determined. post-med, posteromedial; ant, anterior; ant-med, anteromedial; med, medial; post, posterior; lat, lateral; post-lat, posterolateral; ant-lat, anterolateral. III Med on: 19), who had been implanted with the direc- tional Vercise PC (Boston Scientiﬁc) for bilateral STN- DBS between September and December 2015, under- went an extended programming session of their DBS system in the practically deﬁned medication off state (>12 hours of medication withdrawal) 4 to 9 days (mean, 7 6 2) postsurgery. The programming session was sched- uled when the stun effect of electrode placement was decreasing and testable off period motor symptoms had returned in each patient in at least one body side. sequence of levels or horizontal directions tested was left to the programming physician’s discretion, who was unaware of the anatomical position and orienta- tion of the lead within STN. Threshold amplitudes were compiled in a datasheet and later used for con- structing individual polar plots (see Supporting Fig. 2) and descriptive statistics (paired two-sample t test). Patients and Methods Received: 12 February 2016; Revised: 22 March 2016; Accepted: 20 April 2016 Received: 12 February 2016; Revised: 22 March 2016; Accepted: 20 April 2016 Patients and Methods Seven PD patients (2 female; age, 47–64 years; disease duration: 8–20 years; UPDRS-III Med off: 42; UPDRS- Seven PD patients (2 female; age, 47–64 years; disease duration: 8–20 years; UPDRS-III Med off: 42; UPDRS- Published online 31 May 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.26669 Published online 31 May 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.26669 1240 Movement Disorders, Vol. 31, No. 8, 2016 H O R I Z O N T A L C U R R E N T S T E E R I N G I N D B S H O R I Z O N T A L C U R R E N T S T E E R I N G I N D B S TABLE 1. Best and worst effect on TW Most Effective Level Less Effective Level ID Side At level Best DTW (%) Best direction Worst DTW (%) Worst direction At level Best DTW (%) Best direction Worst DTW (%) Worst direction 01 Left 5-6-7 35 post-med –42 post-lat 2-3-4 57 post-med –14 post-lat 01 Right 13-14-15 9 ant –9 post-lat 10-11-12 9 ant –22 post-med 02 Left 5-6-7 28 post-med –48 post-lat 2-3-4 440 post-med –100 post 02 Right 10-11-12 10 ant –50 post-med 13-14-15 100 ant –20 post 03 Right 10-11-12 –6 ant-med –65 post-lat 13-14-15 –3 ant –100 post 04 Right 10-11-12 27 post-med –44 ant-lat 13-14-15 77 post-med 0 ant-lat 05 Left 2-3-4 88 med 0 post-lat 5-6-7 171 ant-med 29 ant 05 Right 13-14-15 –5 post –52 ant-med 10-11-12 135 ant-lat 41 ant 06 Right 13-14-15 –9 lat –75 post-med 10-11-12 47 ant –89 post-med 07 Left 2-3-4 –8 ant-med –72 post-lat 5-6-7 135 post 47 lat 07 Right 13-14-15 9 post –18 post-lat 10-11-12 50 ant-lat 0 post Changes in therapeutic window (DTW) in best and worst direction and respective orientation of electrical ﬁeld vector are given for all STN in which effect and AE threshold could be determined. post-med, posteromedial; ant, anterior; ant-med, anteromedial; med, medial; post, posterior; lat, lateral; post-lat, posterolateral; ant-lat, anterolateral. TABLE 1. Results 1B and 2). The By fusing the postoperative cranial CT with the pre- operative MRI (Leksell Surgiplan; Elekta Instrument AB, Stockholm, Sweden) and identifying the directional lead marker, we determined the orientation of each lead within stereotactic space and assigned each directional setting to an orientation in relation to the anterior com- missure/posterior commissure (AC-PC) line (e.g., anteri- or, anterolateral, etc.). This allowed us to identify the anatomical direction of the current vector providing the 1241 Movement Disorders, Vol. 31, No. 8, 2016 FIG. 1. Bar graph depicting the relative change of the therapeutic window (%) when steering current in the best vs. worst orientation during the monopolar review. Please note, the change in therapeutic window for best directional current steering was proportionally larger at the less effective ring level. On both levels therapeutic windows assessed for the best orientation of current steering differed significantly from those obtained when stimulating into the worst orientation highlighting a potential clinical usefulness of directional DBS. S T E I G E R W A L D E T A L S T E I G E R W A L D E T A L FIG. 1. Bar graph depicting the relative change of the therapeutic window (%) when steering current in the best vs. worst orientation during the monopolar review. Please note, the change in therapeutic window for best directional current steering was proportionally larger at the less effective ring level. On both levels therapeutic windows assessed for the best orientation of current steering differed significantly from those obtained when stimulating into the worst orientation highlighting a potential clinical usefulness of directional DBS. monopolar review in the early postoperative period, they also provide ﬁrst evidence of a beneﬁcial impact of directional DBS on the TW. In theory, a larger TW would offer more programming ﬂexibility for optimizing the efﬁcacy of DBS and reduce the likelihood of inadver- tently exceeding the adverse effect threshold, when the stimulation amplitude is gradually adjusted during the subsequent stabilization period.6 Hence, directional DBS should result in more consistent good outcomes and lower AE rates across groups of patients, whereas it is unlikely to provide more beneﬁt than an optimally implanted ring-mode DBS in an individual. highest positive change in TW (best orientation) and the smallest change (worst orientation) for each lead and level. Results As expected from variable lead locations within a variably shaped and oriented STN, there was no uni- form best direction, and best versus worst orientation were often, but not always strictly opposite (see Table 1). Most often, the “optimal” ﬁeld vector was oriented in an “anterior” or “posteromedial” direction. After grouping the results of directional stimulation by ring level, it became apparent that larger TW effects of optimal current steering could be observed at the less effective (111 6 122%; median, 77; range, –3 to 440) as compared to the most effective level (16 6 22%; median, 9; range, –9 to 88%; see Fig. 1). The notion of a larger TW with directional DBS is also supported by a secondary analysis comparing the amplitude range between efﬁcacy and AE threshold of optimal current steering at the most effective level compared to ring mode (4.0 6 1.5 vs. 3.6 6 1.4 mA; P 5 0.06). Not unexpectedly, we found a larger effect of directional DBS at the less beneﬁcial lead level. This indicates that the individual clinical beneﬁt of directional DBS is best observed for suboptimal electrode positions resulting in a narrow TW, for example, if the electrode is placed too lat- erally within the STN close to the internal capsule. Hence, directional DBS may be able to compensate within certain limits for small deviations of the lead from the optimal functional target, which are a main source of outcome vari- ability in STN-DBS even in experienced surgical centers.4 However, as a note of caution, the availability of a direc- tional DBS system must never be an excuse for lowering the surgical standard and precision of surgical lead place- ment. In fact, the implantation of the Vercise directional lead is surgically more challenging, because the active site has a reduced span, compared to the standard eight- contact ring lead, and the split contacts need to be exactly aligned in depth with the dorsolateral motor region of the STN. Moreover, lead rotation is introduced as an addition- al degree of freedom during the implantation and needs to be controlled for by exact alignment of the rotational lead marker with patient centric landmarks (e.g., AC-PC line). Results Interestingly, the change in TW was driven only by an increase in AE threshold at the most effective level, whereas at the less effective level, both efﬁcacy and AE thresholds, changed signiﬁcantly in favor of the optimal direction (1.25 6 1.23 vs. 1.77 6 0.95 mA; P 5 0.05; 5.39 vs. 4.27 6 1.46 mA; P < 0.005) At the end of the monopolar review, the optimal directional settings were programmed in all patients and gradually adjusted according to clinical needs dur- ing the subsequent days of hospitalization. After a follow-up of 3 to 6 months (median, 4), all patients have remained programmed in directional mode with- out need of rescue programming into ring mode to improve stimulation efﬁcacy (see Supporting Table 1). None of the patients are complaining about stimula- tion induced adverse effects so far. Other limitations of our study include the unblinded and subjective clinical rating of rigidity and adverse effect thresholds, lack of long-term clinical follow-up, early postoperative time period with a partially persis- tent stun effect, and small number of subjects. Impor- tantly, we report feasibility data obtained during an References The lack of a blood-tissue barrier in the appendiceal mucosa would facilitate contact between a blood-borne agent and the enteric nervous system and exogenous agents contacting the host ------------------------------------------------------------ 8. Pollo C, Kaelin-Lang A, Oertel MF. Directional deep brain stimu- lation: an intraoperative double-blind pilot study. Brain 2014; 137(Pt 7):2015-2026. Discussion Our results demonstrate, for the ﬁrst time, the feasibil- ity of horizontal current steering using a fully implanted neurostimulation device with directional leads and MICC technology. Despite the limitations of an acute 1242 Movement Disorders, Vol. 31, No. 8, 2016 A P P E N D E C T O M Y A N D P D acute stimulation challenge, but no efﬁcacy data on the use of chronic directional DBS compared to standard ring DBS. Nevertheless, our ﬁndings may provide valu- able input into the planning of appropriate clinical trials, which are now needed to establish the theoretical advantages of directional DBS in clinical practice and on a group level, but should also take into account possible disadvantages of the expanded param- eter space, such as increased programming burden. ABSTRACT Introduction: Pathogenic movement of alpha-synuclein from the gut to the brain in PD has been proposed. The appendix has a relatively high density of alpha-synuclein deposition in neurologically healthy individuals. We investigated the incidence of PD after appendectomy. Methods: Using cause-speciﬁc hazards regression models, we compared persons over 35 years of age who had undergone appendectomy with two groups of age- and sex-matched individuals having had: (1) a cholecystectomy and (2) neither procedure. Subse- quent diagnoses of PD were identiﬁed. References 1. Deuschl G, Schade-Brittinger C, Krack P, et al. A randomized trial of deep-brain stimulation for Parkinson’s disease. N Engl J Med 2006;355:896-908. 2. Timmermann L, Jain R, Chen L, et al. Multiple-source current steer- ing in subthalamic nucleus deep brain stimulation for Parkinson’s disease (the VANTAGE study): a non-randomised, prospective, mul- ticentre, open-label study. Lancet Neurol 2015;14:693-701. q g Results: Among 42,999 individuals undergoing appendec- tomy, no difference in risk of PD was identiﬁed compared to cholecystectomy (hazard ratio5 1.004; 95% conﬁdence interval: 0.740–1.364). Compared with no procedure, indi- viduals with appendectomy had a higher incidence of PD within 5 years, but no signiﬁcant difference in risk thereafter. Conclusion: In our study, appendectomy in mid or late life does not appear to be associated with a reduced risk of PD. V C 2016 International Parkinson and Move- ment Disorder Society 3. Herzog J, Fietzek U, Hamel W, et al. Most effective stimulation site in subthalamic deep brain stimulation for Parkinson’s disease. Mov Disord 2004;19:1050-1054. 4. Wodarg F, Herzog J, Reese R, et al. Stimulation site within the MRI-deﬁned STN predicts postoperative motor outcome. Mov Dis- ord 2012;27:874-879. 5. Maks CB, Butson CR, Walter BL, Vitek JL, McIntyre CC. Deep brain stimulation activation volumes and their association with neurophysiological mapping and therapeutic outcomes. J Neurol Neurosurg Psychiatry 2009;80:659-666. Key Words: Parkinson’s disease; appendectomy; etiology Key Words: Parkinson’s disease; appendectomy; etiology 6. Volkmann J, Moro E, Pahwa R. Basic algorithms for the program- ming of deep brain stimulation in Parkinson’s disease. Mov Disord 2006;21(Suppl 14):S284-S289. 7. Contarino MF, Bour LJ, Verhagen R, Lourens MA, de Bie RM, van den Munckhof P, Schuurman PR. Directional steering: A novel approach to deep brain stimulation. Neurology 2014;83:1163-1169. It has been proposed that the initiating events of Parkinson’s disease (PD) may occur outside the central nervous system (CNS), with secondary spread to the brain through a prion-like process.1 The gastrointesti- nal tract is a candidate for a location of initiating events.2,3 In people without PD, Gray and colleagues4 found that alpha-synuclein immunoreactivity was most abundant in the appendiceal lamina propria compared to the gastric mucosa or other parts of the right colon, colocalized with neural markers, and was close to the luminal surface of the appendix, putting it in close proximity to any pathogen or triggering event within the gut. Supporting Data Additional Supporting Information may be found in the online version of this article at the publisher’s web-site. Appendectomy in Mid and Later Life and Risk of Parkinson’s Disease: A Population-Based Study Funding agencies: This work was funded by Physician Services Incor- porated. Dr. Austin is supported, in part, by a Career Investigator Award from the Heart and Stroke Foundation (Ontario ofﬁce). Funding agencies: This work was funded by Physician Services Incor- porated. Dr. Austin is supported, in part, by a Career Investigator Award from the Heart and Stroke Foundation (Ontario ofﬁce). 1Morton and Gloria Shulman Movement Disorders Center, Toronto Western Hospital and The Edmond J. Safra Program in PD, Toronto, Ontario, Canada, University of Toronto, Toronto, Ontario, Canada 2The Institute for Clinical Evaluative Sciences and the University of Toronto, Toronto, Ontario, Canada 3Toronto General Research Institute, University Health Network, Department of Surgery, Toronto, Ontario, Canada 4Institute of Health Policy, Management and Evaluation, University of Toronto, Toronto, Ontario, Canada Relevant conﬂicts of interest/ﬁnancial disclosures: Nothing to report. Full ﬁnancial disclosures and author roles may be found in the online ver- sion of this article. Relevant conﬂicts of interest/ﬁnancial disclosures: Nothing to report. Full ﬁnancial disclosures and author roles may be found in the online ver- sion of this article. Received: 27 November 2015; Revised: 5 April 2016; Accepted: 7 April 2016 Published online 31 May 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.26670 Published online 31 May 2016 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/mds.26670 Movement Disorders, Vol. 31, No. 8, 2016 1243 1243 Movement Disorders, Vol. 31, No. 8, 2016
https://openalex.org/W2936237946	https://europepmc.org/articles/pmc6480065?pdf=render	English	null	Fully Biodegradable Composites: Thermal, Flammability, Moisture Absorption and Mechanical Properties of Natural Fibre-Reinforced Composites with Nano-Hydroxyapatite	Materials	2,019	cc-by	7,258	Received: 7 March 2019; Accepted: 1 April 2019; Published: 3 April 2019 Abstract: Natural ﬁbre-reinforced poly(lactic acid) (PLA) laminates were prepared by a conventional ﬁlm stacking method from PLA ﬁlms and natural fabrics with a cross ply layup of [0/90/0/90/0/90], followed by hot compression. Natural ﬁbre (NF) nano-hydroxyapatite (nHA) ﬁlled composites were produced by the same manufacturing technique with matrix ﬁlms that had varying concentrations of nHA in the PLA. Their ﬂammability, thermal, moisture absorption and mechanical properties were analysed in terms of the amount of nHA. The ﬂame behavior of neat PLA and composites evaluated by the UL-94 test demonstrated that only the composite containing the highest quantity of nHA (i.e., 40 wt% nHA in matrix) was found to achieve an FH-1 rating and exhibited no recorded burn rate, whereas other composites obtained only an FH-3. The thermal degradation temperature and mass residue were also observed, via thermogravimetric analysis, to increase when increasing concentrations of nHA were added to the NF composite. The tensile strength, tensile modulus and ﬂexural modulus of the neat resin were found to increase signiﬁcantly with the introduction of ﬂax ﬁbre. Conversely, moisture absorption was found to increase and mechanical properties to decrease with both the presence of NF and increasing concentrations of nHA, and subsequent mechanical properties experienced an obvious reduction. Keywords: poly(lactic acid) (PLA); natural ﬁbre (NF); nano-hydroxyapatite (nHA); ﬂammability; mechanical properties Fully Biodegradable Composites: Thermal, Flammability, Moisture Absorption and Mechanical Properties of Natural Fibre-Reinforced Composites with Nano-Hydroxyapatite Pooria Khalili 1,2 , Xiaoling LIU 1,* , Zirui ZHAO 1 and Brina Blinzler 2 1 Ningbo Nottingham New Materials Research Institute, University of Nottingham Ningbo China (UNNC), Ningbo 315100, China; pooria.khalili@gmail.com (P.K.); zy18541@nottingham.edu.cn (Z.Z.) 2 Department of Industrial and Materials Science, Chalmers University of Technology, 412 96 Gothenburg, Sweden; brina.blinzler@chalmers.se * Correspondence: Xiaoling.Liu@nottingham.edu.cn; Tel.: +86-574-8818-0000 (ext. 8057) Pooria Khalili 1,2 , Xiaoling LIU 1,* , Zirui ZHAO 1 and Brina Blinzler 2 Pooria Khalili 1,2 , Xiaoling LIU 1,* , Zirui ZHAO 1 and Brina Blinzler 2 1 Ningbo Nottingham New Materials Research Institute, University of Nottingham Ningbo China (UNN Ningbo 315100, China; pooria.khalili@gmail.com (P.K.); zy18541@nottingham.edu.cn (Z.Z.) 2 Department of Industrial and Materials Science, Chalmers University of Technology, 412 96 Gothenburg, Sweden; brina.blinzler@chalmers.se * Correspondence: Xiaoling.Liu@nottingham.edu.cn; Tel.: +86-574-8818-0000 (ext. 8057) Pooria Khalili 1,2 , Xiaoling LIU 1,* , Zirui ZHAO 1 and Brina Blinzler 2 1 Ningbo Nottingham New Materials Research Institute, University of Nottingham Ningbo China (UNNC Ningbo 315100, China; pooria.khalili@gmail.com (P.K.); zy18541@nottingham.edu.cn (Z.Z.) 2 Department of Industrial and Materials Science, Chalmers University of Technology, 412 96 Gothenburg, Sweden; brina.blinzler@chalmers.se * Correspondence: Xiaoling.Liu@nottingham.edu.cn; Tel.: +86-574-8818-0000 (ext. 8057) * Correspondence: Xiaoling.Liu@nottingham.edu.cn; Tel.: +86-574-8818-000 materials materials 1. Introduction Polymeric thermosets such as vinyl ester, unsaturated polyester and epoxy are chosen for most composites due to their resistance to chemicals, hydrophobicity and suitable mechanical performances. However, thermoplastic matrices have recently gained signiﬁcant attention in production industries such as construction, automotive and packaging. Bio-based polymers such as thermoplastic starch, poly(lactic acid) (PLA) and poly(3-hydroxybutyrate) (PHB) can be substituted for polyethylene, polystyrene or polypropylene. Petroleum based polymers can be replaced with some bio-based counterparts to deal with disposal issues and problems of diminishing fossil fuel stocks. For many applications where mechanical recycling is an issue, biodegradable polymers are of great interest, as they can be made to be biodegradable and compostable [1]. PLA, amongst other bio-based polymers, possesses relatively high crystallinity, melting point and stiffness, which demonstrates a greater Materials 2019, 12, 1145; doi:10.3390/ma12071145 www.mdpi.com/journal/materials www.mdpi.com/journal/materials Materials 2019, 12, 1145 2 of 13 commercial potential [2,3]. However, for engineering applications, in order to obtain the required mechanical performance, this type of polymer must be reinforced. Recent regulations on the recyclability of materials and environmental requirements compel manufacturers and research institutions to develop composites from renewable sources. Plant ﬁbres (PF) i.e., ﬂax, ramie, jute, hemp and sisal are biodegradable, renewable and economical to use, have high speciﬁc modulus and strength, and have low density [4–6]. PFs possess approximately 40% lower density than glass ﬁbres, which leads to the manufacturing of lighter components than polymeric parts reinforced with glass ﬁbres [7,8]. This is crucial for applications related to transportation resulting in reduced emissions and enhanced fuel efﬁciency [9]. Therefore, the eco-advantages and the reduced mass of these materials make them competitive with synthetic ﬁbre-reinforced composites. However, biocomposites are considered ﬂammable when compared with the traditional ﬁbre-reinforced plastics e.g., carbon and glass ﬁbre composites. Therefore, bio-based composites are more ﬂammable than the traditional composites. This limits the usage of biocomposites in applications where the ﬁre regulations are stringent, for instance the aviation and railway industries [10]. The enhancement of ﬁre and thermal resistivity of biocomposites (e.g., PLA-based biocomposites) is required even for less stringent industries like automotive and packaging. Inorganic ﬁllers such as silica nanoparticles at high concentration were used to impart high thermal stability to the cellulose-based composites, as obtained from thermogravimetric analysis (TGA) [11]. Nano-clay was also used in a different study to improve the thermal stability and ﬂame retardancy of natural ﬁbre polymer composites [12]. 1. Introduction In another investigation, the incorporation of halloysite nanotubes into natural ﬁbre polymeric composites was reported [13]. The thermal degradation temperature was observed to enhance with the incorporation of these nano-ﬁllers. Except for the nano-inorganic additives mentioned, the thermal properties and ﬂame retardancy of natural ﬁbre-reinforced plastics has been improved by the inclusion of various ﬂame-retardants (FRs) into polymers. For instance, the introduction of 3–7 wt% of expandable graphite (EG) [10] and 5–15 wt% ammonium polyphosphate (APP) [6] into 20 wt% natural ﬁbre-reinforced polymer provided 0 s drip ﬂame time from a vertical Bunsen burner test, reduced gross heat of combustion as obtained from bomb calorimeter and increased the mass residue from thermogravimetric analysis. However, the addition of these ﬁllers was observed to reduce the mechanical performance of the FR-ﬁlled composites. This is because high concentrations of inorganic additive, especially compared to organic ﬁllers, are necessary to impart ﬁre resistivity into composites [14]. The high concentrations lead to the embrittlement and deterioration of the mechanical performance of composites. Nano hydroxyapatite (nHA), Ca10(PO4)6(OH)2, is an inorganic bio-ﬁller that has medical applications and is the main calcium phosphate phase present in bone. nHA does not present high strength, which makes it unﬁt for load-bearing applications [15]. Akindoyo et al. [15] showed that addition of 10 wt% nHA into PLA enhanced the content of mass residue from 0.35% to 6.17% at 750 ◦C from thermogravimetric analysis. This demonstrates higher thermal performance of nHA than PLA as inclusion of nHA can result in enhanced char residue. To the best of the authors’ knowledge, few literature studies have reported the improved thermal performance of PLA with the addition of nHA [5,16], and no investigation has highlighted the enhanced ﬂame resistivity and char formation of natural ﬁbre PLA laminates with the inclusion of nHA particles. The water absorption and mechanical behavior of natural ﬁbre-reinforced PLA composites containing nHA additives have also not been reported thus far. The composites were made of fully green materials i.e., bio-based polymer, natural ﬁbre and bio-ﬁller to produce an environmentally friendly composite. In this research, 100% biodegradable composites were developed using various loadings of nHA and these nano-composites were compared with neat PLA and ﬂax ﬁbre-reinforced laminate. The aim of this study was to utilize the good thermal properties of nHA to develop ﬂax fabric PLA composites with improved ﬂame retardancy and thermal resistivity. 2.3. Flammability Test The ﬂame behaviours of the PLA, control and composite laminates were examined using the UL-94 test in accordance with ISO 1210 [17]. The samples were placed horizontally and the burn rate of each sample in the horizontal orientation was reported. The sample is classiﬁed as FH-1 if the combustion front does not pass the 25 mm mark. It is categorized as FH-2 if the combustion front passes the 25 mm mark, but does not pass the 100 mm mark. The burnt length is added to the classiﬁcation designation. It is classiﬁed as FH-3 if combustion front passes the 100 mm mark and the rate of burning does not exceed 75 mm/min for samples measuring a thickness < 3 mm. If the rate of burning surpasses the mentioned value, it is categorized as FH-4. If the ﬂame does not continue after the initial mark, which is 25 mm from the end of specimen, the burn rate is denoted “not applicable” (NA). 2.2. Processing The nanocomposites were compounded using an internal mixer. The PLA along with nHA was compounded at 190 ◦C for 20 min at a mixing speed of 200 rpm. The quantity of nHA was prepared at 20 wt%, 30 wt% and 40 wt% in PLA matrix. The compounding materials (PLA/nHA) and pure PLA were pressed using a hot press machine to produce ﬁlms to be used as the matrix. The ﬁlms with a thickness of roughly 0.3 mm were produced using an XLB50 ( ﬂat vulcanization press (YueQing TOPS machinery CO., Ltd, Shanghai, China) at a pressure of 3 MPa for the period of 10 min at the temperature of 190 ◦C. The PLA and PLA/nHA ﬁlms were cold pressed for 2 min, immediately after removal from the hot press. It is worth noting that the PLA, nHA and ﬁlms were kept in a convectional laboratory oven for 24 h at 50 ◦C before any stage of processing and the ﬂax fabrics were kept in an oven at 80 ◦C prior to the hot compression. Composites were produced by a conventional film-stacking method, which is constructed by adding alternating layers of matrix film and plant fabric. The cross-ply of [0/90/0/90/0/90] was utilized to produce the laminates. The prepared films and flax fabric were hot pressed into 280 × 160 × 2 mm3 plates using the same hydraulic hot press set at a pressure of 5 bar for 10 min at 190 ◦C for consolidation, and subsequently the composite plates were cold pressed to room temperature for 2 min. The prepared composites were named CPLA (control), CN20, CN30 and CN40. CPLA was made of flax fabric and PLA, and the number in front of CN20, CN30 and CN40 (nano-composites) denotes the amount of nHA in the matrix. The CPLA and nano-composites contained 30% fibre volume fraction. Neat PLA was also fabricated for comparison purposes. 1. Introduction The formulations produced were investigated based on ﬂammability, thermal, mechanical and water absorption behaviors. 3 of 13 Materials 2019, 12, 1145 2.1. Materials Unidirectional ﬂax fabrics were purchased from Easy Composites Ltd (Stoke-on-Trent UK) and had the mass over area and density of 150 g/m2 and 1.5 g/m3, respectively. Nano-hydroxyapatite (nHA), Ca10(PO4)6(OH)2 ≥99.5%, was supplied by Yunduan New Materials, Weifang, China. The particle size was less than 40 nm, had the density of 3.16 g/cm3 and had pH of 7.41. The mass loss after drying was 0.59% and mass loss after burning was 2.59%. The poly (lactic acid) (PLA, IngeoTM bio-based polymer PLA3251D) was provided by NatureWorks LLC (Minnetonka, MN, USA) and had the speciﬁc gravity of 1.24 g/cm3. 2.5. Differential Scanning Calorimeter (DSC) Test 2.5. Differential Scanning Calorimeter (DSC) Test The differential scanning calorimeter (DSC) test was conducted on a TA machine. (SDT Q20) (New Castle, DE, USA, 2016). Samples weighing 17–19 mg were heated from room temperature (25 ◦C) to 200 ◦C at a ramping rate of 10 ◦C/min with a nitrogen ﬂow rate of 50 mL/min. The same heating rate and ﬂow rate were used to cool down the samples to the room temperature. The crystallinity of PLA based matrix was obtained using the Equation (1) [18] below: XDSC% = ∆Hm −∆Hc ∆H ◦ m × 100 w (1) (1) where ∆Hm is the melting enthalpy, ∆Hc is the enthalpy of cold crystallization, ∆H ◦ m is 93.7 J/g for pure crystalline PLA and w is the mass fraction of the PLA-based matrix. 2.6. Scanning Electron Microscopy (SEM) 2.6. Scanning Electron Microscopy (SEM) Scanning electron microscopy (SEM, ZEISS Sigma/VP SEM) (ZEISS IGMA/VF, Jena, Germany, 2012) was used to investigate the morphological structure of fractured surfaces of composites after tensile experiments with an acceleration voltage of 5 kV. A Leica EM SCD 500 high vacuum Sputter Coater (Lecia Microsystems, Prague, Czech Republic, 2012) was employed to gold coat the fracture surfaces with the plasma exposition of 60 s prior to scanning. 2.4. Thermogravimetric Analysis (TGA) Thermogravimetric analysis (TGA) was conducted on a TA instrument (SDT Q600) (New Castle, DE, USA, 2016) to investigate the thermal stability of the composites. The samples of approximately 20 mg were placed in a platinum crucible and were heated from 30 ◦C to 600 ◦C in a nitrogen environment. The ﬂow rate and ramping rate were set to 50 mL/min and 20 ◦C/min, respectively. The corresponding TGA and differential thermal gravimetry (DTG) were obtained Materials 2019, 12, 1145 4 of 13 2.5. Differential Scanning Calorimeter (DSC) Test 2.7. Tensile Test The tensile test was carried out on dog bone specimens prepared according to EN ISO 527-4 [19], using an MTS EXCEED E45 universal tester (MTS Systems Corporation, Shanghai, China 2016). The specimens were conditioned at 50% relative humidity and 23 ◦C prior to the testing at the crosshead speed of 1 mm/min. The extensometers were used in the middle of gauge length. The average results of ﬁve specimens were recorded for the tensile strength, tensile modulus and elongation at break. 2.8. Flexural Test The bending test was conducted according to EN ISO 14125 [20] on an MTS ECCEED E42 universal tester (MTS Systems Corporation, Shanghai, China) at a speed of 0.5 mm/min. The specimens measured 60 mm × 15 mm × 2 mm with the span length of 40 mm and were preconditioned the environmental method mentioned in the previous test. The ﬂexural strength and modulus were recorded as an average of the ﬁve specimens. 3. Results 3.1. Flammability of Poly(lactic Acid) (PLA), Control and Nano-Hydroxyapatite (nHA)-Filled Compos ability of Poly(lactic Acid) (PLA), Control and Nano-Hydroxyapatite (nHA)-Filled Composites The UL-94 results are displayed based on the rate of burning and rating in Table 1. The PLA sample burned continuously until its burn length reached 31 mm and sample drips fell on the chamber bed. For the Control, CN20 and CN30, the UL-94 rating was FH-3, indicating the complete combustion of these composites. The burn rates were recorded 20, 19.6 and 17.7 mm/min, respectively, and no dripping was observed. This demonstrates that increasing volumes of nHA enhanced the ﬂame retardancy. Further addition of nHA (CN40) improved the ﬁre resistivity signiﬁcantly by providing self-extinguishing performance and no burn rate. This is due to the formation of a more thermally stable char in the respective sample, thereby acting as an effective barrier, shielding the underlying materials from the ﬂame zone and heat. PLA undergoes thermal decomposition via a hydroxyl end-initiated ester interchange process and chain hemolysis creating lactide, oligomers, carbon dioxide, carbon monoxide and acetaldehyde [19], and ﬁllers that can act as ﬂame-retardants are capable of transforming its decomposition pathway to produce char and decrease the formation of combustible products [22,23]. Table 1. UL-94 results of poly(lactic acid) (PLA), Control and nano-hydroxyapatite (nHA) ﬁlled composite laminates. Table 1. UL-94 results of poly(lactic acid) (PLA), Control and nano-hydroxyapatite (nHA) ﬁlled composite laminates. Sample Burn Rate (mm/min) UL 94 Rating PLA NA FH-2-31 mm CPLA 20 FH-3 CN20 19.6 FH-3 CN30 17.7 FH-3 CN40 NA FH-1 3.2. Thermal Studies 2.9. Water Absorption Test The water absorption test was conducted for all the formulations in accordance with EN ISO 62:2008 [21]. Prior to the test, the samples were conditioned at room temperature with 50% relative humidity after being dried in an oven for 72 h at 40 ◦C. The specimens were then soaked in two different containers ﬁlled with distilled water at the temperatures of 25 ◦C and 60 ◦C. The amount of moisture absorption was measured per 24 h for 14 days. m0 is the mass of test sample after initial drying and before immersion, and m is the mass of test sample after immersion and ﬁnal drying. The average mass gain of three test samples for each formulation was calculated and reported. The mass gain (c) is the percentage change in mass relative to the initial mass and is calculated using the Equation (2): c = m −m0 m0 × 100% (2) c = m −m0 m0 × 100% (2) 5 of 13 Materials 2019, 12, 1145 3.2. Thermal Studies The thermogravimetric analysis (TGA) and differential thermogravimetric analysis (DTGA) curves of the composite samples are shown in Figures 1 and 2. The degradation temperatures, mass residue and mass loss rate of the samples were determined. The TGA curve displayed one decomposition step for all formulations and that the main decomposition took place between 300 ◦C and 400 ◦C. It is worth noting that bio-ﬁbres consist of hemicellulose, lignin and cellulose, and their pyrolysis takes place at different temperature ranges of approximately 160–900 ◦C, 220–315 ◦C and 315–400 ◦C for lignin, hemicellulose and cellulose, respectively [24,25]. The temperatures at 5% mass loss (T5%) of PLA, CPLA and nanocomposites were approximately 328 ◦C, 304 ◦C and 290 ◦C, and the degradation temperatures (Td), obtained from the peak of DTGA curves, were about 373 ◦C, 361 ◦C and 372 ◦C, respectively. This demonstrates that the T5% and Td of CPLA and nHA ﬁlled composites are lower than those of PLA, which can be explained by a higher destabilization of PLA in the composites. Amongst the composites, the Td improved with the addition of nHA relative to that of the Control and revealed nearly the same value as neat PLA. It was reported that electrostatic attraction between the polymeric carboxylate group and CA2+ of nHA ions affect the interfacial bonding in composites containing nHA [26]. The maximum rates of thermal degradation (Figure 2) were measured 2.88, 1.94, 1.91, 1.7 and 1.67 %/◦C for PLA, CPLA, CN20, CN30 and CN40, respectively, suggesting that both the natural ﬁbre and nHA contributed to the reduction in the decomposition rate. The mass residues at 600 ◦C (Figure 1) were 1.85%, 8.14%, 16.97%, 22.70% and 30.84% for PLA, CPLA, CN20, CN30 and CN40, respectively. The inclusion of natural ﬁbres was observed to improve the mass reside by about 340 % relative to that of neat PLA, and introduction of 40 wt% nHA into the matrix was found to increase the residue by 279% compared to that of CPLA. This enhancement in the increased char residue is in accordance with ﬂammability test, which resulted in the formation of thermally resistive char on the surface of CN40 and subsequent protection of the bulk of the substrate. 6 of 13 Materials 2019, 12, 1145 Figure 1. Thermogravimetric analysis (TGA) curves of PLA, control and nanocomposites. Figure 1. Thermogravimetric analysis (TGA) curves of PLA, control and nanocomposites. Figure 2. 3.2. Thermal Studies Differential thermogravimetric analysis (DTGA) curves of PLA, control and nanocomposites. 3. Crystallization and Melting Properties Figure 2. Differential thermogravimetric analysis (DTGA) curves of PLA, control and nanocomposites. Figure 2. Differential thermogravimetric analysis (DTGA) curves of PLA, control and nanocomposites. 3.3. Crystallization and Melting Properties Figure 2. Differential thermogravimetric analysis (DTGA) curves of PLA, control and nanocomposites. 3.3. Crystallization and Melting Properties The DSC thermograms of CPLA, PLA and CN40 from the heating run are displayed in Figure 3. The glass transition temperature (Tg), crystallization temperature (Tc), melting temperature (Tm), crystallization enthalpy (∆Hc), melting enthalpy (∆Hm) and degree of crystallinity (XDSC) obtained are shown in Table 2. The addition of natural ﬁbres was observed to show an increase in Tg compared to that of PLA. This observation displays that an increased Tg consequently indicates a change from ﬂexible and soft behaviors to tough and hard properties [27]. The inclusion of nHA slightly decreased the Tg, indicating improved polymer chain mobility. The crystallinity and Tc of CPLA decreased by 12 ◦C after incorporation of ﬁbres, which implies that the natural ﬁbres hinder the diffusion and migration of molecular chains of PLA to the nucleus surface in CPLA. As expected, the addition of nHA was observed to further reduce the Tc by approximately 13%, signifying a faster crystallization of the nanocomposites. This can be ascribed to nHA acting as sites of nucleation, leading to heterogeneous nucleation within the PLA [28]. A similar increase in Tg (by approximately. 2 ◦C) and reduction in Tc (by about 14 ◦C) was obtained after the addition of talc ﬁbre into the PLA [29]. For the last transition, the formation of a smaller second peak is due to the presence of NFs in the PLA which inﬂuenced the overall melting behavior of the composites. This suggests the presence of two different types of crystal [30]. Inducing heterogeneous nucleation due to the introduction of nHA can also contribute to the higher Tm. This is attributed to the creation of less perfect crystals, which would usually melt at greater temperatures than more perfect crystals [15]. 7 of 13 Materials 2019, 12, 1145 Figure 3. Differential scanning calorimeter (DSC) thermograms of PLA, control (CPLA) and CN40. Figure 3. Differential scanning calorimeter (DSC) thermograms of PLA, control (CPLA) and CN40. Table 2. The Tg, Tc, Tm, ∆Hc, ∆Hm and XDSC obtained from DSC test. 3.2. Thermal Studies Samples Tg (◦C) Tc (◦C) Tm (◦C) ∆Hc (J/g) ∆Hm (J/g) XDSC (%) PLA 57 118 150 6.02 9.04 4.95 CPLA 59 106 148 17.47 18.41 1.54 CN40 56 92 151 13.83 15.04 1.98 3.4. Morphological Properties The fracture surface of the control and composites containing nHA particles were scanned after tensile tests were performed, as illustrated in Figure 4. As observed from Figure 4a, no ﬁbre pull-out or obvious evidence of poor interfacial bonding in the ﬂax ﬁbre-reinforced PLA can be seen. The red and yellow arrows highlight the ﬂax ﬁbres and PLA matrix in the composite, respectively. The impact of the inclusion of nHA ﬁllers in the composite laminates (CN20, CN30 and CN40) can be observed in their breaking behavior, as shown in Figure 4b–d. A smooth fracture surface was observed in the matrix of CPLA, whereas a rough fracture surface was obtained in the matrix of the nanocomposites. Incorporation of 20 wt% nHA into the matrix was detected to be sufﬁcient to provide even dispersion of particles in the PLA-based matrix, as depicted in the red circles Figure 4b. However, some ﬁbre pull-out and ﬁbre debonding were observed, indicating poor ﬁbre/matrix interfacial adhesion. As the amount of ﬁller increased in the matrix, clear agglomeration was found in the composite laminates ((Figure 4c,d), which is displayed in yellow circles. The agglomerated spots can be points where stress concentration occurs, which can cause premature failure of the composites. It has been stated [28] that well-distributed nHA ﬁller in the PLA matrix can result in improved mechanical performances. Materials 2019, 12, 1145 8 of 13 8 of 13 Figure 4. Scanning electron microscope (SEM) micrographs of the fracture surface of tensile tested samples at 500× magniﬁcation. (a) CPLA, (b) CN20, (c) CN30 and (d) CN40. Figure 4. Scanning electron microscope (SEM) micrographs of the fracture surface of tensile tested samples at 500× magniﬁcation. (a) CPLA, (b) CN20, (c) CN30 and (d) CN40. Figure 4. Scanning electron microscope (SEM) micrographs of the fracture surface of tensile tested samples at 500× magniﬁcation. (a) CPLA, (b) CN20, (c) CN30 and (d) CN40. 3.5. Tensile Properties The tensile properties of the PLA, control and nHA loaded laminates were studied and the results are illustrated in Figure 5. The Young’s moduli of the PLA and Control were 5.1 GPa and 12.6 GPa, the tensile strengths, 45 MPa and 50.7 MPa, and the elongation at break 4.5% and 2%, respectively. As expected, reinforcing the PLA with 6 layers of ﬂax fabric enhanced the tensile strength and modulus by 13% and 146% compared to neat PLA, respectively, which is a signiﬁcant improvement. This is because tensile performance is primarily ﬁbre-dependent and ﬂax ﬁbre has greater tensile properties than those of neat PLA. Moreover, the observed enhancements in mechanical performance are affected by good interfacial adhesion between matrix and ﬁbre, as demonstrated in Figure 4a, leading to better load transfer between ﬁbre and matrix, and thereby improved mechanical performance. However, after the addition of fabrics, the elongation at break experienced a reduction. As compared to the control, the tensile properties decreased with the inclusion of nHA due to ﬁbre pull-out and ﬁbre debonding. The reduction was substantial upon the addition of 30 wt% and 40 wt% nHA in the matrix, which can be attributed to the low strength of nHA as revealed previously [26,31]. Its low strength can be associated with the elimination of organic compounds during synthesis [15]. Another reason could be the agglomeration of nHA in the matrix, as revealed by the SEM micrographs (Figure 4c,d), which triggers an early failure. 9 of 13 Materials 2019, 12, 1145 Figure 5. Tensile properties of PLA, control and nHA ﬁlled composites. Figure 5. Tensile properties of PLA, control and nHA ﬁlled composites. 3.6. Flexural Properties The ﬂexural performances of the PLA, control and nHA loaded laminates were investigated, and the results are displayed in Figure 6. Neat PLA had a ﬂexural strength and modulus of 57.5 MPa and 2.9 GPa, respectively, whereas the control showed a slightly lower bending strength of 54 MPa and a signiﬁcantly higher modulus of 9.1 GPa than those of neat PLA, indicating 212% improvement in ﬂexural modulus. As observed in Figure 6, both the ﬂexural strength and the modulus reduced with the increased nHA as compared to those of the control. For instance, for the highest amount of nHA (CN40), the reduction in strength and modulus was 67% and 35%, respectively, relative to those of the control. As mentioned in Section 3.5, the drop can be related to the low strength of nHA, which occurred during its synthesis because of elimination of organic components. The second reason for these reductions is the issue of nHA dispersion in the PLA matrix. Poor dispersion results in agglomeration (Figure 4c,d) which affects the ﬂexural properties as they are matrix dependent. Signiﬁcant agglomeration can result in unwanted premature failure at the interface of PLA and nHA as well as ineffective load transfer. Figure 6. Flexural properties of PLA, control and nHA ﬁlled composites. Figure 6. Flexural properties of PLA, control and nHA ﬁlled composites. 3.7. Water Absorption Behaviors After 11 days, the mass of the samples started reducing slightly, which was due to the separation of tiny PLA pieces as a result of disintegration of the water-soluble materials [32]. The CN20, CN30 and CN40 samples saturated rapidly, after 4, 2 and 2 days, when compared to the saturation time obtained at room temperature, respectively. The mass gain was higher for the samples with greater concentration of nHA. These composites experienced a continuous mass loss after the peak of mass gain due to peeling and degradation of the nHA/PLA samples. This could be due to the poor interfacial adhesion between the nHA/PLA matrix and the ﬂax ﬁbre, thereby creating a higher surface area for degradation of the matrix material. It is worth highlighting that more cracks were visibly be observed on the surface of composites reinforced with ﬂax ﬁbre than neat PLA, and the cracks were more obvious for composites containing higher concentrations of nHA. gain occurred for two consecutive days and reached approximately 0.6% at the saturation point. The mass remained almost constant for the following days. With the addition of natural fabric into the PLA (CPLA composite), the mass gain increased up to 12 days resulting in a higher saturation value of 8.9%. This is attributed to the hydrophilic nature of ﬂax ﬁbre due to the polar groups e.g., carboxyl and hydroxyl groups. The results obtained are in agreement with another report [32], suggesting that hydrophobic PLA demonstrates a lower tendency to absorb water than lignocellulosic fabrics [33]. For CN20 and CN30, the mass gain continued for 10 days and reached 16.3% and 16.7%, respectively. The saturation time for CN40 was 6 days with the mass gain of 19.8%. This proves that addition of nHA ﬁllers into the matrix increased the water absorption of ﬂax ﬁbre composites, indicating poor interfacial adhesion between the ﬁbres and matrix due to the inclusion of nano ﬁllers, as detected in SEM images (Figure 4), and demonstrated in mechanical analysis. This is because increasing the concentration of nano ﬁllers reduces the amount of PLA in the composite. As a result, less matrix is available to adhere to the natural ﬁbre, thereby exposing the ﬁbres to the environment. Therefore, the natural ﬁbres absorbed more water due to their lack of adhesion to the PLA/nHA matrix. In addition, nHA is a polar (hydrophilic) ﬁller, which further increases the water absorption tendency. 3.7. Water Absorption Behaviors The mass gain as a function of the immersion time (day) at 25 ◦C and 60 ◦C for the produced composites is displayed in Figures 7 and 8. For PLA at room temperature (Figure 7), the mass 10 of 13 Materials 2019, 12, 1145 gain occurred for two consecutive days and reached approximately 0.6% at the saturation point. The mass remained almost constant for the following days. With the addition of natural fabric into the PLA (CPLA composite), the mass gain increased up to 12 days resulting in a higher saturation value of 8.9%. This is attributed to the hydrophilic nature of ﬂax ﬁbre due to the polar groups e.g., carboxyl and hydroxyl groups. The results obtained are in agreement with another report [32], suggesting that hydrophobic PLA demonstrates a lower tendency to absorb water than lignocellulosic fabrics [33]. For CN20 and CN30, the mass gain continued for 10 days and reached 16.3% and 16.7%, respectively. The saturation time for CN40 was 6 days with the mass gain of 19.8%. This proves that addition of nHA ﬁllers into the matrix increased the water absorption of ﬂax ﬁbre composites, indicating poor interfacial adhesion between the ﬁbres and matrix due to the inclusion of nano ﬁllers, as detected in SEM images (Figure 4), and demonstrated in mechanical analysis. This is because increasing the concentration of nano ﬁllers reduces the amount of PLA in the composite. As a result, less matrix is available to adhere to the natural ﬁbre, thereby exposing the ﬁbres to the environment. Therefore, the natural ﬁbres absorbed more water due to their lack of adhesion to the PLA/nHA matrix. In addition, nHA is a polar (hydrophilic) ﬁller, which further increases the water absorption tendency. The water absorption behavior of composites immersed in water at 60 ◦C is displayed in Figure 8. The PLA reached the peak of its mass gain with the value of ca. 0.8% and it became saturated rapidly after about one day. This shows higher amount of mass gain and shorter saturation time as compared to those of PLA at room temperature. For CPLA, the mass gain increased for around 11 days and its value was recorded at 10.4%. These results also show a higher mass gain and shorter saturation relative to those of CPLA soaked at room temperature. 3.7. Water Absorption Behaviors , p ( y p ) , p y The water absorption behavior of composites immersed in water at 60 ◦C is displayed in Figure 8. The PLA reached the peak of its mass gain with the value of ca. 0.8% and it became saturated rapidly after about one day. This shows higher amount of mass gain and shorter saturation time as compared to those of PLA at room temperature. For CPLA, the mass gain increased for around 11 days and its value was recorded at 10.4%. These results also show a higher mass gain and shorter saturation relative to those of CPLA soaked at room temperature. After 11 days, the mass of the samples started reducing slightly, which was due to the separation of tiny PLA pieces as a result of disintegration of the water-soluble materials [32]. The CN20, CN30 and CN40 samples saturated rapidly, after 4, 2 and 2 days, when compared to the saturation time obtained at room temperature, respectively. The mass gain was higher for the samples with greater concentration of nHA. These composites experienced a continuous mass loss after the peak of mass gain due to peeling and degradation of the nHA/PLA samples. This could be due to the poor interfacial adhesion between the nHA/PLA matrix and the ﬂax ﬁbre, thereby creating a higher surface area for degradation of the matrix material. It is worth highlighting that more cracks were visibly be observed on the surface of composites reinforced with ﬂax ﬁbre than neat PLA, and the cracks were more obvious for composites containing higher concentrations of nHA. Figure 7. Water absorption behavior of PLA, CPLA and nanocomposites at 23 ◦C. Figure 7. Water absorption behavior of PLA, CPLA and nanocomposites at 23 ◦C. 11 of 13 Materials 2019, 12, 1145 Figure 8. Water absorption behavior of PLA, CPLA and nanocomposites at 60 ◦C. Figure 8. Water absorption behavior of PLA, CPLA and nanocomposites at 60 ◦C. Conﬂicts of Interest: The authors declare no conﬂict of interest. 4. Conclusions This research studied environmentally friendly biocomposites which were prepared using hot compression with the incorporation of fully bio-sourced constituents (i.e., bio-ﬁller, NF and bio-thermoplastic). The study demonstrates the ﬂame resistivity and thermal improvement of nHA as an additive by preventing ﬂame development in the composite specimen (CN40) and reducing the burn rate as obtained via the UL-94 test study. It should also be noted that the mass residue was enhanced by 279%, thermal decomposition temperature was improved slightly, and the mass loss rate was reduced by approximately 14% upon the addition of the nanoﬁllers determined by the initial TGA investigation. The efﬁciencies were more pronounced in terms of ﬂame retardancy and thermal resistivity at higher concentrations of nHA particles. From the DSC analysis, it was found that the degree of crystallinity reduced in CPLA when compared to that of neat PLA, and with the presence of nHA additives in the composite, XDSC experienced an increase as compared to its value of CPLA. The nHA particles, while imparting good thermal resistivity to the composite laminates, induced lower mechanical properties at higher concentrations. This reduction could be attributed to the agglomeration of the nHA particles within the matrix, in particular at higher concentrations, as well as increased ﬁbre pull-out/debonding upon nHA inclusion, based on morphological observations. The water absorption increased with the addition of NF as well as nHA particles at both room temperature and 60 ◦C, which is ascribed to the poor adhesion at the interface between nHA/PLA matrix and ﬂax ﬁbres, as validated in the SEM micrograph, thereby enabling easier moisture absorption of NFs in the composites. This is because there is less matrix available to coat the natural ﬁbres, which results in easy exposure to the environment. Furthermore, water absorption tendency increases in the presence of inorganic ﬁllers such as nHA. At higher temperature, the saturation time was shortened remarkably for all the composites. In addition, the matrixof composite specimens started to disintegrate throughout the test due to the hydrolytic degradation. Therefore, there must be a compromise between the ﬂame retardancy/thermal resistivity and mechanical/moisture absorption behavior for these types of biocomposites. Author Contributions: Conceptualization, P.K.; Funding acquisition, X.L.; Investigation, P.K.; Methodology, P.K. and Z.Z.; Project administration, X.L.; Resources, X.L.; Supervision, P.K.; Validation, X.L. and B.B.; Writing original draft, P.K.; Review and editing, X.L. and B.B. Funding: This work was done in “the ACC TECH-UNNC joint laboratory in Sustainable Composite Materials”. The authors would like to acknowledge the ﬁnancial support by Ningbo S&T bureau collaboration project (project code: 2017D10030) and the Ningbo 3315 Innovation Team, Scheme of “Marine Composites Development and Manufacturing for Sustainable Environment. Author Contributions: Conceptualization, P.K.; Funding acquisition, X.L.; Investigation, P.K.; Methodology, P.K. and Z.Z.; Project administration, X.L.; Resources, X.L.; Supervision, P.K.; Validation, X.L. and B.B.; Writing original draft, P.K.; Review and editing, X.L. and B.B. References 2008, retardants for natural ﬁber reinforced epoxy composite. Compos. Part B Eng. 2017, 114, 101–110. [CrossRef] 7. Shen, L.; Patel, M.K. Life Cycle Assessment of Polysaccharide Materials: A Review. J. Polym. Environ. 2008, 16, 154. [CrossRef] 7. Shen, L.; Patel, M.K. Life Cycle Assessment of Polysaccharide Materials: A Review. J. Polym. Environ. 2008, 16, 154. [CrossRef] 8. Corbière-Nicollier, T.; Gfeller Laban, B.; Lundquist, L.; Leterrier, Y.; Månson, J.A.E.; Jolliet, O. Life cycle assessment of bioﬁbres replacing glass ﬁbres as reinforcement in plastics. Res. Conserv. Recycl. 2001, 33, 267–287. [CrossRef] 9. John, M.J.; Thomas, S. Bioﬁbres and biocomposites. Carbohydr. Polym. 2008, 71, 343–364. [CrossRef] 10. Khalili, P.; Tshai, K.Y.; Kong, I. Natural ﬁbre reinforced expandable graphite ﬁlled composites: Evaluation of the ﬂame retardancy, thermal and mechanical performances. Compos. Part A Appl. Sci. Manuf. 2017, 100, 194–205. [CrossRef] 11. Rodríguez-Robledo, M.C.; González-Lozano, M.A.; Ponce-Peña, P.; Quintana Owen, P.; Aguilar-González, M.A.; Nieto-Castañeda, G.; Bazán-Mora, E.; López-Martínez, R.; Ramírez-Galicia, G.; Poisot, M. Cellulose-Silica Nanocomposites of High Reinforcing Content with Fungi Decay Resistance by One-Pot Synthesis. Materials 2018, 11, 575. [CrossRef] 12. Biswal, M.; Mohanty, S.; Nayak, S.K. Thermal stability and ﬂammability of banana-ﬁber-reinforced polypropylene nanocomposites. J. Appl. Polym. Sci. 2012, 125, E432–E443. [CrossRef] 13. Subasinghe, A.; Das, R.; Bhattacharyya, D. Materials, N. Study of thermal, ﬂammability and mechanical properties of intumescent ﬂame retardant PP/kenaf nanocomposites. Int. J. Smart Nano Mater. 2016, 7, 202–220. [CrossRef] 14. Hapuarachchi, T.; Ren, G.; Fan, M.; Hogg, P.; Peijs, T. Fire retardancy of natural ﬁbre reinforced sheet moulding compound. Appl. Compos. Mater. 2007, 14, 251–264. [CrossRef] 15. Akindoyo, J.O.; Beg, M.D.; Ghazali, S.; Heim, H.P.; Feldmann, M. Effects of surface modiﬁcation on dispersion, mechanical, thermal and dynamic mechanical properties of injection molded PLA-hydroxyapatite composites. Compos. Part A Appl. Sci. Manuf. 2017, 103, 96–105. [CrossRef] 16. Akindoyo, J.O.; Beg, M.D.; Ghazali, S.; Heim, H.P.; Feldmann, M. Impact modiﬁed PLA-hydroxyapatite composites–Thermo-mechanical properties. Compos. Part A Appl. Sci. Manuf. 2018, 107, 326–333. [CrossRef] 17. ISO. Thermal Properties. In Method 140A: Determination of the Burning Behaviour of Horizontal and Vertical Specimens in Contact with a Small-Flame Ignition Source; International Organization for Standardization: Geneva, Switzerland, 1992; Vol. BS 2782-1: Method 140A: 1992 ISO 1210:1992. 18. Pilla, S.; Gong, S.; O’Neill, E.; Rowell, R.M.; Krzysik, A.M. Polylactide-pine wood ﬂour composites. Polym. Eng. Sci. 2008, 48, 578–587. [CrossRef] 19. ISO. Plastics—Determination of Tensile Properties. 4. Conclusions Funding: This work was done in “the ACC TECH-UNNC joint laboratory in Sustainable Composite Materials”. The authors would like to acknowledge the ﬁnancial support by Ningbo S&T bureau collaboration project (project code: 2017D10030) and the Ningbo 3315 Innovation Team, Scheme of “Marine Composites Development and Manufacturing for Sustainable Environment. 12 of 13 12 of 13 Materials 2019, 12, 1145 References 1. 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W2993379694.txt	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/DD3CF9D4FF665BFB0339597C22CF5FE2/S2047102519000256a.pdf/div-class-title-domestic-courts-and-the-paris-agreement-s-climate-goals-the-need-for-a-comparative-approach-div.pdf	en		Domestic Courts and the Paris Agreement's Climate Goals: The Need for a Comparative Approach	Transnational environmental law	2,019	cc-by	9,105	Transnational Environmental Law, 9:1 (2020), pp. 37–54 © 2019 The Author(s) This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/ licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited. doi:10.1017/S2047102519000256 SYMPOSIUM ARTICLE Domestic Courts and the Paris Agreement’s Climate Goals: The Need for a Comparative Approach† Anna-Julia Saiger* First published online 5 December 2019 Abstract Domestic courts enjoy generous attention in international political and legal climate change literature. As a result of the reluctance of national governments to pursue climate protection measures, courts are called on to enforce international climate goals. This article assesses two domestic climate change cases (the Thabametsi Case and the Vienna Airport Case) in the light of Anthea Roberts’ functional understanding of the role of domestic courts in international law. It argues that domestic courts play a pivotal role in linking international obligations of conduct with national obligations of result. This role depends on domestic contexts and, therefore, requires a comparative approach. Keywords: Climate change litigation, Comparative approach, Thabametsi Case, Vienna Airport Case, Domestic courts in international law 1.  The current international climate change regime draws increasing scholarly interest towards domestic measures that translate international goals into concrete actions and legal decisions at the national and subnational levels. The existing legal literature documents how domestic courts play a prominent role among the diverse actors of the multi-layered climate change regime. This is as a result of a signiﬁcant number of strategic climate change cases before domestic judiciaries. Civil society and individuals all over the world sue their governments in order to bring about more ambitious climate † * This contribution is part of a collection of articles growing out of the conference ‘Climate Change Litigation’ held at Aarhus University Department of Law, Aarhus (Denmark), 14–15 June 2018. Humboldt University, Berlin (Germany). Email: saigeran@hu-berlin.de. The author is grateful to Katerina Mitkidis and the anonymous TEL reviewers for valuable comments on the article. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 38 Transnational Environmental Law, 9:1 (2020), pp. 37–54 protection measures or use legal remedies to oblige corporations to pay compensation for harm caused by climate change.1 The claimants seek to bolster their legal arguments by lobbying for the interpretation of national climate change policies and laws in accordance with states’ international legal obligations under the United Nations Framework Convention on Climate Change (UNFCCC)2 and the 2015 Paris Agreement.3 While the role of domestic courts in international law has been characterized by their ability to ‘bring international law home’,4 the current climate change regime illustrates how complex this role may be. By combining top-down with bottom-up elements, the Paris Agreement5 questions the ability of domestic courts to ‘wear two hats’6 – i.e., to enforce and, at the same time, to ‘create’7 international law. Under the Paris Agreement each state is obliged to formulate its own mitigation commitments at the national level.8 This provision relates to the long-term temperature stabilization goal, but it does not require the parties to hold ‘the increase in the global average temperature to well below 2°C above pre-industrial levels’.9 In relation to this objective, the Paris Agreement establishes an obligation of conduct, not an obligation of result.10 If at all, such obligations of result may be found within national legal systems. Domestic constitutional and administrative provisions, inter alia, play a pivotal role in this respect. Because of the dynamic interplay between international obligations and national legal systems, climate change cases that refer to the Paris Agreement are pertinent for examining the role of domestic courts in international law and discussing the methodological requirements for such an undertaking. Anthea Roberts suggests viewing domestic courts as enforcers and creators of international law.11 To grasp 1 2 3 4 5 6 7 8 9 10 11 J. Setzer & R. Byrnes, ‘Global Trends in Climate Change Litigation: 2019 Snapshot’, 4 July 2019, Grantham Research Institute on Climate Change and the Environment and Centre for Climate Change Economics and Policy, London School of Economics and Political Science, available at: http://www. lse.ac.uk/GranthamInstitute/publication/global-trends-in-climate-change-litigation-2019-snapshot; M. Burger & J. Gundlach, ‘The Status of Climate Change Litigation: A Global Review’, May 2017, United Nations Environment Program and Sabin Center for Climate Change Law, available at: http://columbiaclimatelaw.com/ﬁles/2017/05/Burger-Gundlach-2017-05-UN-Envt-CC-Litigation.pdf. New York, NY (US), 9 May 1992, in force 21 Mar. 1994, available at: https://unfccc.int/resource/docs/ convkp/conveng.pdf. Paris (France), 12 Dec. 2015, in force 4 Nov. 2016, available at: http://unfccc.int/paris_agreement/items/ 9485.php. H.H. Koh, ‘1998 Frankel Lecture: Bringing International Law Home’, Faculty Scholarship Series, Paper 2102, pp. 623–81, available at: https://digitalcommons.law.yale.edu/cgi/viewcontent.cgi?article=2883& context=fss_papers. Art. 2(1)(a), Paris Agreement. A. Roberts, ‘Comparative International Law? The Role of National Courts in Creating and Enforcing International Law’ (2011) 60(1) International & Comparative Law Quarterly, pp. 57–92, at 59. Ibid. Art. 4(2), Paris Agreement: ‘Each Party shall prepare, communicate and maintain successive nationally determined contributions that it intends to achieve. Parties shall pursue domestic mitigation measures, with the aim of achieving the objectives of such contributions’. Art. 2(1)(a), Paris Agreement. L. Rajamani, ‘Ambition and Differentiation in the 2015 Paris Agreement: Interpretative Possibilities and Underlying Politics’ (2016) 65(2) International & Comparative Law Quarterly, pp. 493–514, at 497. Roberts, n. 6 above. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 39 the ‘co-constitutive process’12 of law enforcement and law creation, domestic climate change cases should be approached using a comparative method. Roberts and her co-authors deﬁne comparative law as a means to ‘identify, analyz[e], and explain similarities and differences in the interpretation and application of international law’.13 Departing from the common frame of international law, comparative analyses allow for an understanding of the domestication of international norms in different jurisdictions. Thus far, most of the literature assessing domestic climate change litigation and its importance for the international legal regime focuses on the ability of the courts to enforce international law. The literature follows a governance approach to domestic climate change cases. However, the cases are ‘not about litigants seeking to enforce the Paris Agreement domestically, but testing domestic policies enacted to give effect to international commitments’.14 The governance approach, therefore, does not provide the appropriate methodological tools to assess the role of domestic courts within the bottom-up approach of the Paris Agreement. Within the regulatory structure of the Agreement the international and domestic levels merge into a hybrid interplay of procedural and substantive obligations. Therefore, the role of domestic courts presupposes a comparative understanding of both the international obligations and the national contexts in which the litigation takes place. What are the requirements for such a comparative analysis and what can it tell us about the role of domestic courts within the regulatory bottom-up approach of the international climate change regime? This article challenges the scholarly account of the role of domestic courts in international climate change law. It calls for a functional understanding based on Roberts and connects this claim with the methodological prerequisites for a comparative approach that considers national contexts. Section 2 presents the debate on the role of domestic courts in international law from George Scelle’s functional approach to current discussions about the need for and the requirements of comparative analyses within international law. The debate illustrates the development of the double-sided function advocated by Roberts, placing the courts in a dynamic relationship between international and national law. Section 3 aligns this debate with the role assigned to domestic courts in the international climate change regime. It reveals the governance bias underlying many scholarly contributions. According to the literature in this ﬁeld, rather than fulﬁlling a hybrid function of law enforcement and law creation, domestic courts are considered actors in the global governance ‘kaleidoscope’15 of climate change mitigation. 12 13 14 15 M.A. Waters, ‘Mediating Norms and Identity: The Role of Transnational Judicial Dialogue in Creating and Enforcing International Law’ (2005) 93(2) Georgetown Law Journal, pp. 487–574, at 502 et seq. A. Roberts et al., ‘Conceptualizing Comparative International Law’, in A. Roberts et al. (eds), Comparative International Law (Oxford University Press, 2018), pp. 3–31, at 7 (emphasis in original). K. Bouwer, ‘The Unsexy Future of Climate Change Litigation’ (2018) 30(3) Journal of Environmental Law, pp. 483–506, at 492. Bouwer, ibid., p. 492. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 40 Transnational Environmental Law, 9:1 (2020), pp. 37–54 Section 4 assesses the role of the courts based on two domestic climate change cases. The South African Thabametsi Case16 and the Austrian Vienna Airport Case17 both question the signiﬁcance of the Paris Agreement’s climate goals within the decisionmaking processes of the construction of carbon-intensive projects. The courts refer quite differently to the Paris Agreement’s long-term temperature stabilization goal and the steps taken by the legislators in the form of the nationally determined contributions (NDCs). The hybrid function of law enforcement and law creation is so closely connected to and dependent on the national legal system that it gives rise to the question how international law can make sense of domestic judicial decisions. Therefore, Section 5 proposes a comparative approach in order to understand the hybrid role of domestic courts in the international climate change regime since the Paris Agreement. This article transposes Roberts’ differentiation of the double-sided function fulﬁlled by domestic courts within international law into the realm of international climate change law. However, the bottom-up approach of the Paris Agreement adds another layer to the process of law enforcement and law creation. The role of domestic courts in enforcing and creating international law is determined by their ability to link international obligations of conduct with national obligations of result. This ability depends on national legal provisions and contexts and on the role of courts within their own domestic legal systems. 2.         In an early contribution on the role of domestic courts in the interplay of international and domestic law, George Scelle developed the notion of the dédoublement fonctionnel. Once they deal with international provisions, domestic courts act as international courts. Formally, the courts are part of their domestic legal system but, at the same time, they function as the third force in international law.18 Scelle supported a federal vision of international law and, ultimately, advocated the establishment of international courts.19 For Scelle, the role of domestic courts as international adjudicative bodies was of only a provisional character. The subsequent literature followed this functional approach, transplanting it into a dualist vision of international law. According to this line of thinking, domestic courts help to ﬁll the enforcement gap within international law.20 While, in Scelle’s thinking, 16 17 18 19 20 Earthlife Africa v. Minister of Environmental Affairs, High Court of South Africa, 65662/16, 8 Mar. 2017 (Thabametsi Case). Vienna Airport Case, Federal Administrative Court (BVwG Wien), W109 2000179-1/291E, 2 Feb. 2017; Constitutional Court (VfGH), E 875/2017-32, E 886/2017-31, 29 June 2017. G. Scelle, Précis de droit des gens: Principes et systématique, Vol. 1: ‘Introduction, Le milieu intersocial’ (Recueil Sirey, 1932), p. 56. Note that the dédoublement fonctionnel applies to all three branches of government: G. Scelle, ‘Le Phénomène juridique du dédoublement fonctionnel’, in W. Schätzel & H.J. Schlochauer (eds), Rechtsfragen der internationalen Organisation: Festschrift für Hans Wehberg zu seinem 70, Geburtstag (Klostermann, 1956), pp. 324–42, at 331. B. Conforti, ‘National Courts and the International Law of Human Rights’, in B. Conforti & F. Francioni (eds), Enforcing International Human Rights in Domestic Courts (Brill/Nijhoff, 1997), pp. 3–14; R.A. Falk, ‘The Role of Domestic Courts in the International Legal Order’ (1964) 39(3) Indiana Law Journal, pp. 429–45. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 41 the universality of international law is unavoidable (international law prevails over national law),21 domestic courts now become part of the fragile process of implementing international law into national legal systems. From this new perspective scholars were able to question the conditions that allow domestic courts to enforce international law. Their independence from the executive is among the most important factors.22 However, until the 1990s the number of domestic courts engaging with international law was still very limited.23 References to international law by domestic courts have become more common in recent times.24 Based on this body of cases, the literature called for a descriptive analysis of the jurisprudence. Antonio Cassese asked for an examination of the motivations underlying various judicial decisions.25 About two decades later, Eyal Benvenisti revealed that courts mainly follow domestic institutional considerations when applying international law.26 He stressed that courts use international law to counterbalance the weight of the executive in a globalized world.27 This shift towards legal realism not only views courts as enforcers of international law but concurrently examines their role in the process of fragmentation of international norms. It vests courts with a lawmaking function.28 Hence, the effectiveness as well as the unity of international law is at stake. Two methodological pathways have been suggested for assessing the role of domestic courts in effectuating and determining international law: international relations, and comparative international law. The former method takes a governance approach to the role of domestic courts. As international problems become global, they necessitate a broad range of actors to play a role in problem solving. Human rights violations, cartels within global markets, and climate change rank among those problems.29 The literature views courts as actors of global governance and examines their ability to fulﬁl this role. At the same time, scholars emphasize the legal, political, and cultural contexts within which courts act.30 They reveal the tension between a universal aspiration underlying international provisions and their domestic realization. Karen Knop speaks 21 22 23 24 25 26 27 28 29 30 Scelle, n. 18 above, p. 31. B. Conforti, ‘Notes on the Relationship between International Law and National Law’ (2001) 3(1) International Law Forum du Droit International, pp. 18–24, at 21. E. Benvenisti, ‘Comments on the Systemic Vision of National Courts as Part of an International Rule of Law’ (2012) 4(1) Jerusalem Review of Legal Studies, pp. 42–49, at 48. Y. Shany, ‘Dédoublement Fonctionnel and the Mixed Loyalties of National and International Judges’, in F. Fontanelli, G. Martinico & P. Carrozza (eds), Shaping Rule of Law through Dialogue: International and Supranational Experiences (Europa Law, 2010), pp. 27–44, at 32, 33, 36. A. Cassese, ‘Remarks on Scelle’s Theory of “Role Splitting” (dédoublement fonctionnel) in International Law’ (1990) 1(1) European Journal of International Law, pp. 210–31, at 219. E. Benvenisti, ‘Reclaiming Democracy: The Strategic Uses of Foreign and International Law by National Courts’ (2008) 102(2) American Journal of International Law, pp. 241–74, at 242. Benvenisti, n. 26 above, pp. 245 et seq. Roberts, n. 6 above, p. 68. R. Michaels, ‘Global Problems in Domestic Courts’, in S. Muller et al. (eds), The Law of the Future and the Future of the Law (Torkel Opsahl Academic EPublisher, 2011), pp. 165–75, at 167. H.G. Schermers, ‘The Role of Domestic Courts in Effectuating International Law’ (1990) 3(3) Leiden Journal of International Law, pp. 77–85, at 79 et seq.; H. Aust, ‘The Rules of Interpretation as Secondary Rules: The Perspective of Domestic Courts’, in M. Heupel & T. Reinold (eds), The Rule of https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 42 Transnational Environmental Law, 9:1 (2020), pp. 37–54 about the perception of domestic courts as ‘translators’ of international law in the following terms: ‘Just as we know that translation from one language to another requires more than literalness, we must recognize the creativity, and therefore the uncertainty, involved in domestic interpretation’.31 This metaphor accepts the complex interplay of contexts that determine the role of domestic courts. Building on Knop, Roberts calls for a comparative approach to examine the role of domestic courts: Instead of seeing national court decisions through the prism of whether they enforce or breach international law, the comparative international law lens focuses our attention on the way in which domestic courts nationalize substantive international law in diverse ways, resulting in a hybridity that is ripe for comparative analysis.32 Within this approach, both directions of inﬂuence (from the international level to the national level and vice versa) may be taken into scholarly account. This ‘microcomparison’33 allows for a cultural understanding of the jurisprudence34 while at the same time recognizing the dual role of domestic courts in enforcing and creating international law. 3.       There is an extensive body of literature on the role of domestic courts in the international climate change regime.35 However, the contributions refer only rarely to the 31 32 33 34 35 Law in Global Governance (Palgrave, 2016), pp. 66 et seq.; A. Nollkaemper, National Courts and the International Rule of Law (Oxford University Press, 2011), p. 10. K. Knop, ‘Here and There: International Law in Domestic Courts’ (1999/2000) 32(2) NYU Journal of International Law & Politics, pp. 501–35, at 506. Roberts, n. 6 above, p. 74. Ibid., p. 60. D.W. Kennedy, ‘New Approaches to Comparative Law: Comparativism and International Governance’ (1997) 2 Utah Law Review, pp. 545–673, at 554. See, e.g., H.M. Osofsky, ‘Climate Change Litigation as Pluralist Legal Dialogue’ (2007) 43A(1) Stanford Journal of International Law, pp. 181–237; E.A. Posner, ‘Climate Change and International Human Rights Litigation: A Critical Appraisal’ (2007) 155(6) University of Pennsylvania Law Review, pp. 1925–45; W.C. Burns & H.M. Osofsky (eds), Adjudicating Climate Change: State, National, and International Approaches (Cambridge University Press, 2009); C. Hilson, ‘Climate Change Litigation in the UK: An Explanatory Approach (or Bringing Grievance Back In)’, in F. Fracchia & M. Occhiena (eds), Climate Change: La Risposta del Diritto (Editoriale Scientiﬁca, 2010), pp. 421–36; J. Lin, ‘Climate Change and the Courts’ (2012) 32(1) Legal Studies, pp. 35–57; D. Markell & J.B. Ruhl, ‘An Empirical Assessment of Climate Change in the Courts: A New Jurisprudence or Business as Usual?’ (2012) 64(1) Florida Law Review, pp. 15–86; J. Peel, L. Godden & R.J. Keenan, ‘Climate Change Law in an Era of Multi-Level Governance’ (2012) 2(1) Transnational Environmental Law, pp. 245–80; J. Peel & H.M. Osofsky, Climate Change Litigation: Regulatory Pathways to Cleaner Energy (Cambridge University Press, 2015); M. Wilensky, ‘Climate Change in the Courts: An Assessment of Non-U.S. Climate Litigation’ (2015) 26(1) Duke Environmental Law & Policy Forum, pp. 131–79; R. Abate, Climate Justice: Case Studies in Global and Regional Governance Challenges (Environmental Law Institute, 2016); B. Preston, ‘The Contribution of Courts in Tackling Climate Change’ (2016) 28(1) Journal of Environmental Law, pp. 11–7; A. Averchenkova, S. Frankhauser & M. Nachmany (eds), Trends in Climate Change Legislation (Edward Elgar, 2017). For a literature review see J. Setzer & L.C. Vanhala, ‘Climate Change Litigation: A Review of Research on Courts and Litigants in Climate Governance’ (2019) WIREs Climate Change, pp. 1–19. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 43 methodological debate in international legal scholarship concerning the role of domestic courts in international law.36 Instead, they focus on the ability of domestic courts to contribute to the global governance challenge of climate change.37 As Elizabeth Fisher states, ‘[ j]udicial reasoning is of less importance than the actual bringing of the litigation and the outcome of such litigation’.38 The underlying motivation of many scholarly contributions is to solve the complex governance problem of climate change. Litigation is viewed through the lens of possible responses to institutional failures within other branches of government.39 In this regard the successful ﬁrst instance Urgenda decision in 2015 sets an ‘example for the world’.40 Scholars emphasize the effects of domestic court decisions on global governance challenges. Scholarly accounts of climate change litigation and adjudication follow the multi-layered governance perspective established in earlier literature.41 The roles attributed to courts are varied and numerous. They ﬁll legal gaps between international objectives and national policies, put pressure on governments and corporations, and inﬂuence public discourses.42 Courts also realize climate justice in individual cases,43 give legal meaning to the provisions of the Paris Agreement44 and ensure that governments are held accountable.45 Courts stress the urgency of policy changes,46 and develop and promote climate change governance.47 To illustrate this point, it is helpful to take a look at the literature advocating a sceptical stance towards climate change litigation. For example, in his 2007 contribution Eric Posner questions the ability of the courts to contribute to climate protection.48 36 37 38 39 40 41 42 43 44 45 46 47 48 For an exception see P.G. Ferreira, ‘“Common But Differentiated Responsibilities” in the National Courts: Lessons from Urgenda v. The Netherlands’ (2016) 5(2) Transnational Environmental Law, pp. 329–51, at 332 et seq. (showing that there has been a comparative discussion in international environmental law but not in international climate change law). An earlier exception is the reference to A.M. Slaughter in Osofsky, n. 35 above, p. 191 et seq. Bouwer, n. 14 above, p. 493. E. Fisher, ‘Climate Change Litigation, Obsession and Expertise: Reﬂecting on the Scholarly Response to Massachusetts v. EPA’ (2013) 35(3) Law & Policy, pp. 236–60, at 241. Ibid., p. 240 et seq. R. Cox, ‘A Climate Change Litigation Precedent: Urgenda Foundation v The State of the Netherlands’ (2016) 34(2) Journal of Energy & Natural Resources Law, pp. 143–63, at 161. See also J. van Zeben, ‘Establishing a Governmental Duty of Care for Climate Change Mitigation: Will Urgenda Turn the Tide?’ (2015) 4(2) Transnational Environmental Law, pp. 339–57; B. Mayer, ‘The State of the Netherlands v. Urgenda Foundation: Ruling of the Court of Appeal of The Hague (9 October 2018)’ (2019) 8(1) Transnational Environmental Law, pp. 167–92. Peel, Godden & Keenan, n. 35 above, pp. 249, 251 (who draw on D. Bodansky & E. Diringer, as well as on E. Ostrom). Peel & Osofsky, n. 35 above, p. 16. Abate, n. 35 above, pp. 1006, 1010. D. Estrin, ‘Limiting Dangerous Climate Change: The Critical Role of Citizen Suits and Domestic Courts – Despite the Paris Agreement’, CIGI Papers, No. 101, May 2016, pp. 2–26, at 5, available at: https://www.cigionline.org/sites/default/ﬁles/paper_no.101.pdf. Lord Carnwath JSC, ‘Climate Change Adjudication after Paris: A Reﬂection’ (2016) 28(1) Journal of Environmental Law, pp. 5–9, at 9. Peel, Godden & Keenan, n. 35 above, p. 271. Preston, n. 35 above, pp. 13, 15. Posner (n. 35 above) starts his article by asking: ‘What is the appropriate legal and political strategy for limiting the emission of greenhouse gases?’: ibid., p. 1925. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 44 Transnational Environmental Law, 9:1 (2020), pp. 37–54 Jacqueline Peel, Lee Godden and Rodney Keenan point towards procedural barriers and view courts as mediators acting indirectly on climate governance.49 Others point to the high volume of unsuccessful cases50 and warn against backlashes.51 All of these contributions share the initial governance perspective on domestic courts in the climate change regime and ‘emphasize the actors over the structures’.52 There are several reasons for the focus on domestic courts as actors in global climate change governance. It has been stated that the literature pays particular attention to certain ‘high proﬁle climate change cases’.53 One of the ﬁrst examples is the academic discussion of Massachusetts v. EPA in 2007.54 At that time, the Kyoto Protocol was about to enter into force.55 Its ﬁrst commitment period spanned 2008 to 2012. The United States (US) never ratiﬁed the Kyoto Protocol and was therefore bound only by the general objectives of the UNFCCC. Domestic courts became new fora in which climate protection efforts could be pursued: In the US, the refusal of the Bush Administration (January 2001–January 2009) to undertake climate change regulation under existing environmental laws or to support the promulgation of any new climate change laws caused deep frustration and undeniably prompted action through the courts to put pressure on the executive branch to act on climate change.56 The search for alternative climate protection drivers was intensiﬁed by the role of the US as a major emitter of greenhouse gases (GHGs); this also holds true for Australia.57 Moreover, most climate change litigation has taken place in these two jurisdictions. Accordingly, the literature predominantly assesses these two jurisdictions. From its inception the debate was closely focused on common law systems. The deadlock in international negotiations in relation to the second commitment period of the Kyoto Protocol, from 2009 in Copenhagen to 2012 in Doha, led to a lack of conﬁdence in international law.58 At the same time, a growing body of climate change litigation in other jurisdictions provoked scholarly responses. The governance approach to domestic courts thus expanded to other jurisdictions.59 49 50 51 52 53 54 55 56 57 58 59 Peel, Godden & Keenan, n. 35 above, p. 272; Lin, n. 35 above, p. 39. S. Varvaštian, ‘Climate Change Litigation, Liability and Global Climate Governance: Can Judicial Policy-making Become a Game-changer?’, Berlin Conference ‘Transformative Global Climate Governance après Paris’, May 2016, pp. 1–8, at 4, 5, available at: https://refubium.fu-berlin.de/bit stream/handle/fub188/18585/Varvastianxclimatexchangexlitigation.pdf?sequence=1&isAllowed=y. M. Miller, ‘The Right Issue, the Wrong Branch: Arguments Against Adjudicating Climate Change Nuisance Claims’ (2010) 109(2) Michigan Law Review, pp. 257–89, at 283. M. Heupel & T. Reinold, ‘Introduction: The Rule of Law in an Era of Multi-level Governance and Global Legal Pluralism’, in Heupel & Reinold (eds), n. 30 above, pp. 1–25, at 5. Bouwer, n. 14 above, p. 489. Massachusetts v. EPA, US Supreme Court, 127 S.Ct. 1438, 1446–47, 2 April 2007. Kyoto Protocol to the United Nations Framework Convention on Climate Change, Kyoto (Japan), 10 Dec. 1997, in force 16 Feb. 2005, available at: http://unfccc.int/kyoto_protocol/items/2830.php. Lin, n. 35 above, p. 37; see also Posner, n. 35 above, p. 1944. Peel & Osofsky, n. 35 above, p. 3. Ibid., p. 10. Wilensky, n. 35 above, pp. 175 et seq. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 45 However helpful in assessing governance dynamics regarding climate change, this approach falls short in fully grasping the role of domestic courts within the bottom-up approach of the Paris Agreement. National procedural and substantive rules determine the ability of courts to enforce or create international law. The national provenance of contributions to climate change mitigation requires scholarly attention to understand national circumstances in depth. The following two case studies serve as examples which assess the hybrid function of law enforcement and law creation fulﬁlled by domestic courts in light of the Paris Agreement’s regulatory approach. 4.   The South African Thabametsi Case60 and the Austrian Vienna Airport Case61 have been chosen as they are embedded in very different legal systems and contexts but deal with a similar legal question. In both cases the court considered the international climate goals of the Paris Agreement within domestic administrative provisions. Domestic courts were called to decide on the duty of the competent authorities to interpret administrative law in light of the Paris Agreement’s long-term temperature stabilization goal. Both cases illustrate the hybrid function the courts fulﬁl within the regulatory bottom-up approach of the Paris Agreement. They reveal the complexity of this role and the need for a methodological toolset to understand the domestic contexts within the international legal architecture. 4.1. South Africa: Earthlife Africa v. Minister of Environmental Affairs In the Thabametsi Case, Earthlife Africa Johannesburg, an environmental nongovernmental organization (NGO), challenged the authorization of a coal-ﬁred power plant in the Limpopo Province in northern South Africa. As is the case today, the country faced adverse climate change impacts in the form of extreme weather events and water scarcity. Even so, the national energy policy increased South Africa’s reliance on coal.62 At the centre of the case was the National Environmental Management Act (NEMA).63 According to section 24 NEMA an environmental authorization must precede the construction of a power plant. The Act states that ‘the potential consequences for or impacts on the environment of listed activities … must be considered, investigated, assessed and reported on to the competent authority’.64 On 25 February 2015 60 61 62 63 64 Thabametsi Case, n. 16 above. Vienna Airport Case, n. 17 above. Integrated Research Plan for Electricity 2010–2030, available at: http://www.energy.gov.za/ﬁles/irp_ frame.html; Thabametsi Case, n. 16 above, p. 11, paras 25, 26; T.-L. Humby, ‘The Thabametsi Case: Case No 65662/16 Earthlife Africa Johannesburg v Minister of Environmental Affairs’ (2018) 30(1) Journal of Environmental Law, pp. 145–55, at 145; J.C. Ashukem, ‘Setting the Scene for Climate Change Litigation in South Africa: Earthlife Africa Johannesburg v Minister of Environmental Affairs and Others [2017] ZAGPPHC 58 (2017) 65662/16’ (2017) 13(1) Law, Environment and Development Journal, pp. 35–43, at 41. National Environmental Management Act, No. 107 (1998), Government Gazette, No. 19519, 27 Nov. 1998. Ibid., s. 24(1). https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 46 Transnational Environmental Law, 9:1 (2020), pp. 37–54 Thabametsi, the eponymous operator, obtained an environmental authorization from the Chief Director of the Department of Environmental Affairs (DEA). Earthlife challenged this decision, ﬁrstly, before the Minister and, secondly, together with the Minister’s appeal decision, before the Gauteng High Court in Pretoria. The challenge was based on the ground that, according to the NEMA, the environmental impact assessment (EIA) must consist of ‘all relevant factors’ relating to the environment.65 Interpreted in the light of domestic66 and international provisions,67 climate change impacts would constitute one such relevant factor. The claimant stressed that the environmental authorization violated the NEMA and should be set aside because the authorities had failed to assess and consider climate change impacts.68 Earthlife asked for a new decision based on a climate change impact assessment. The DEA, as well as Thabametsi as an interested party, argued that there was no express provision to include climate change in the EIA under either domestic or international law. The assessment and consideration of climate change impacts could not constitute a mandatory requirement. They stressed that it was at the government’s discretion to decide on climate change measures, as such measures were always to be balanced against South Africa’s development needs. The Thabametsi power plant would ensure that the country’s energy demands could be met. In her appeal decision, the Minister accepted the need for a climate change impact assessment but nonetheless upheld the authorization. To her decision she added an amendment obliging Thabametsi to prepare a climate change report. In its ruling of 8 March 2017 the High Court reviewed the decisions of the Chief Director and the Minister and asked whether there was a duty to consider climate change impacts resulting from section 24-O(1) NEMA. The court took a purposive approach towards the provisions covering EIA and related them to the interpretative principles of the NEMA: namely, sustainable development and the precautionary principle.69 It tied the interpretation to section 24 of the Constitution of South Africa,70 granting each citizen the right to a clean environment.71 Moreover, under section 223 of the Constitution, priority is given to any interpretation which is in line with international obligations. The court acknowledged the international legal obligations to consider climate change impacts in national policies,72 but also stressed that the international legal regime allows for the development of coal-ﬁred power plants in the immediate future.73 South Africa’s NDC envisages a peak in GHG emissions up 65 66 67 68 69 70 71 72 73 Ibid. Environmental Impact Assessment Regulations, Government Gazette, No. 33306, 18 June 2010; s. 24 Constitution of the Republic of South Africa, 8 May 1996, amended 11 Oct. 1996. The UNFCCC and the Paris Agreement. Promotion of Administrative Justice Act (PAJA) of 2000, s. 6(2)(e)(iii), Government Gazette, No. 20853, 3 Feb. 2000. Thabametsi Case, n. 16 above, para 80, p. 32. Constitution of the Republic of South Africa, n. 66 above. Thabametsi Case, n. 16 above, paras 80, 81, pp. 32, 33. Ibid., para. 83, pp. 33, 34. Ibid., para. 35, pp. 14, 15. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 47 to 2020. Thus, its reliance on coal is anticipated to decrease over a longer timescale. According to the court, the consideration of climate change impacts was necessary to correspond with this peak, plateau, and decline trajectory stipulated by the NDC.74 The second question was whether the duty to consider climate change impacts was fulﬁlled. The court deduced from an interpretation of the Minister’s appeal decision that this was not the case. The absence of a climate change impact assessment (admitted by the Minister’s amendment to the initial authorization) prevented the Chief Director from balancing all relevant factors.75 A general consideration of climate change at the national level would not sufﬁce; the impacts must be assessed speciﬁcally in relation to the project in question.76 Citing the claimants’ representative, the court pronounced that ‘[i]t is simply impossible to strike an appropriate equilibrium where the details of one of the key factors to be balanced are not available to the decision-maker’.77 With regard to the appeal decision, the court concluded that the Minister ought to substitute the initial decision with her own decision based on the climate change impact assessment.78 Finally, the appeal decision was set aside by the court, obliging the Minister to issue a new decision. The court afﬁrmed the duty of the administrative decision maker to consider climate change impacts, but this left room for the authorities to weigh up development needs against climate protection.79 Accordingly, the Minister’s decision, newly issued after the judgment, granted the construction permit for the power plant even in the light of the climate change impacts subsequently assessed.80 4.2. Austria: Vienna Airport Case The Vienna Airport Case addressed a similar legal question. In this case, the construction of the third runway of Vienna Airport was challenged before the courts. After a lengthy EIA procedure,81 the government of Lower Austria, as the competent EIA authority, issued the construction permit in 2012.82 According to section 3(3) of the EIA Act (2000)83 this decision included consideration of all relevant legal provisions. If one of them was not met, the permit could not be issued (concentrated procedure).84 Citizens’ initiatives, neighbours, and the city of Vienna appealed against the decision to the newly established Federal Administrative Court. They argued that the authorities 74 75 76 77 78 79 80 81 82 83 84 Ashukem, n. 62 above, pp. 40, 42. Thabametsi Case, n. 16 above, para. 100, p. 39. Ibid., para. 95, p. 37. Ibid., para. 100, pp. 39, 40. NEMA, n. 63 above, s. 43. Thabametsi Case, n. 16 above, para. 35, p. 14. The High Court states: ‘South Africa’s international obligations anticipate and permit the development of new coal ﬁred stations in the immediate term’. Humby, n. 62 above, p. 155. This decision is currently being challenged in a new appeal procedure. The application was handed in on 1 Mar. 2007 following a mediation procedure from 2001 to 2005: see Vienna Airport Case (BVwG), n. 17 above, para. I.1.2., pp. 5, 6. Government of Lower Austria, Authorization RU4-U-302/301-2012, 10 July 2012, Vienna Airport Case, (BVwG), n. 17 above, para. I.2., p. 8. Federal Environmental Impact Assessment Act 2000, BGBl. No. 697/1993, 14 Oct. 1993. G. Kirchengast et al., ‘BVerwG versagt Genehmigung wegen überwiegenden Interesses am Klimaschutz’ (2017) 3 Recht der Umwelt, pp. 121–31, at 121. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 48 Transnational Environmental Law, 9:1 (2020), pp. 37–54 had unlawfully failed to consider the climate change impacts of the project. According to section 71(1) and (2) of the Austrian Aviation Act,85 the public interests opposing the construction may not outweigh the public interests in having the third runway. On 2 February 2017, the ﬁrst instance Federal Administrative Court decided on the interpretation of those ‘other public interests’. The Aviation Act itself does not provide any criteria by which to assess which interests must be considered and how they are to be balanced. The court, therefore, referred to the hierarchy of the Austrian legal system and to the actions taken by democratically elected branches of government.86 Austria’s international obligations under the UNFCCC and the Paris Agreement would oblige the authorities to integrate climate change impacts into their balancing of interests in the decision-making procedure of major projects. Austria envisages sectoral emissions ceilings in its 2012 Climate Protection Act, which was revised in 2017.87 This Act transposes Austria’s obligation to reduce GHG emissions in sectors that are not part of the emissions trading scheme by 16% in relation to the 2005 baseline until 2020. As a European Union (EU) Member State, Austria did not submit its own NDC under the Paris Agreement, but committed to the EU’s target of a 40% reduction in GHG emissions by 2030 in relation to 1990 baselines.88 Importantly, the court not only reviewed the administrative decision but, in accordance with section 28(2) of the Federal Act on Proceedings of Administrative Courts,89 substituted the decision. This occurs whenever the initial authorization is unlawful on the ground of a lack of consideration of all relevant circumstances in the decisionmaking process at the administrative level.90 Based on a 128-page assessment, the court concluded that the opposing ‘other public interests’ – namely, climate change impacts – outweigh the public interests in favour of the construction of the runway. It withdrew the construction permit. According to the Austrian Constitutional Court, which issued its judgment on 29 June 2017, the reasoning of the Federal Administrative Court was unconstitutionally arbitrary. Only six months after the ﬁrst instance decision, the Constitutional Court concluded that the UNFCCC and the Paris Agreement were not applicable to the case. It stated that in referring to international obligations in its reasoning, the Federal Administrative Court had severely misjudged the legal meaning of those norms.91 The Constitutional Court concluded that the decision of the Federal Administrative Court violated the claimants’ right to equality before the law. 85 86 87 88 89 90 91 Federal Aviation Act, BGBl. No. 253/1957, 12 Dec. 1957. Vienna Airport Case (BVwG), n. 17 above, para. III.4.5.14., p. 121; A.J. Saiger, ‘Climate Change Protection Goes Local: Remarks on the Vienna Airport Case’, Verfassungsblog, 20 Mar. 2017, available at: https://verfassungsblog.de/climate-change-protection-goes-local-remarks-on-the-vienna-airport-case. Federal Act on Climate Protection (KSG), BGBl. I No. 106/2011, 21 Nov. 2011. Intended Nationally Determined Contribution of the EU and Its Member States, 6 Mar. 2015, para. 3, p. 1, available at: https://www4.unfccc.int/sites/ndcstaging/Pages/Home.aspx. See Kirchengast et al., n. 84 above, p. 129. Federal Act on Proceedings of Administrative Courts (VwVG), BGBl. I No. 33/2013, 13 Feb. 2013. Kirchengast et al., n. 84 above, p. 122; Vienna Airport Case (BVwG), n. 17 above, para. III.4.5.2., p. 113. G. Kirchengast et al., ‘VfGH behebt Untersagung der dritten Piste’ (2017) 6 Recht der Umwelt, pp. 252–64, at 253. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 49 While the Constitutional Court agreed on the need to carry out a balancing of interests, it underlined that the speciﬁc public interests were to be found in the Aviation Act itself.92 The Paris Agreement would not apply at the domestic level, as it generates only international obligations.93 Political statements by democratically elected branches of government could not be treated as law. Constitutional norms with a focus on sustainability94 could guide the interpretation of provisions of the Aviation Act only where environmental protection was already foreseen. If this were the case, the Constitution would reinforce the environmental goods protected by the provisions in question.95 As the Aviation Act (which entered into force in 1957) did not refer to environmental protection, the Constitution could not guide the interpretation of indeterminate legal norms of this Act. The Constitutional Court found that by including the ‘cruise-emissions’96 in the calculation of the adverse climate impacts caused by the third runway, the ﬁrst instance court did not respect Austrian territory as the ultimate reference for the assessment of emissions and their impacts on the climate.97 On the basis of this reasoning, the Constitutional Court annulled the ﬁrst instance decision and maintained the construction permit of the third runway. 4.3. The Hybrid Function of Law Enforcement and Law Creation The two case studies will be assessed against the backdrop of Roberts’ understanding of domestic courts as law enforcers and law creators in relation to international law. In its present form this hybrid function raises the question whether public authorities ought to consider the climate goals of the Paris Agreement in their domestic decision-making processes, and what their signiﬁcance is within national legal systems. Both functions are closely interconnected.98 Roberts points towards the danger of discussants labelling judicial decisions the outcome of which they support as examples of impartial law enforcement, whereas unwelcome decisions are cast as instances of partial law creation.99 The bottom-up approach of the Paris Agreement renders it even more difﬁcult to deduce from international law whether one court correctly enforces international legal provisions whereas another incorrectly fails to do so and, therefore, attributes a partial interpretation of international provisions. As the applicability and substantive meaning of the Paris Agreement’s climate goals depend on national legal provisions, this question may be answered only in light of national law. 92 93 94 95 96 97 98 99 Vienna Airport Case (VfGH), n. 17 above, para. 204, p. 59. Ibid., para. 213, p. 62. Federal Constitutional Act on Sustainability, Animal Protection, Comprehensive Environmental Protection, Water and Food Supply Safety and Research, BGBl. I No. 111/2013, 11 July 2013. Kirchengast et al., n. 91 above, p. 258. ‘Cruise emissions’ encompass the whole ﬂight, not just emissions from landing and take-off (LTO emissions). Vienna Airport Case (VfGH), n. 17 above, para. 204, p. 59. A. Tzanakopoulos, ‘Domestic Courts in International Law: The Judicial Function of National Courts’ (2011) 34 Loyola Los Angeles International & Comparative Law Review, pp. 133–68, at 135. Roberts, n. 6 above, p. 61. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 50 Transnational Environmental Law, 9:1 (2020), pp. 37–54 The case studies show that the reference to the Paris Agreement’s climate goals necessitates a constellation within the domestic legal system that allows for consideration of international law. None of the claimants directly asked for enforcement of the Paris Agreement’s long-term temperature stabilization goal. This can be explained by the character of Articles 2(1) and 4(2) of the Paris Agreement as an obligation of conduct. Rather, the question was whether domestic administrative provisions should be interpreted in a way that includes the Paris Agreement’s climate goals. In the Thabametsi Case, the ‘potential consequences for or impacts on the environment’ that needed to be assessed according to the NEMA invited the court to apply an interpretation that is in line with international legal provisions and, accordingly, to integrate consideration of climate change impacts into this domestic provision. In the Vienna Airport Case, the ‘other public interests’ referred to in section 71(1) and (2) of the Aviation Act led the ﬁrst instance court to refer to the Paris Agreement. The court saw a lacuna within the Aviation Act. It did not give any guidance on how to interpret the indeterminate legal provision at stake. The court, therefore, referred to other legal provisions, such as the Paris Agreement, for its interpretation. The Constitutional Court, however, took the opposite approach and argued that the reference to climate goals was not possible as long as there was no concrete provision in the Aviation Act that allowed for or required such an interpretive exercise. The Constitutional Court stated that the Paris Agreement, as an international instrument, generated mere international obligations and could not apply within national law.100 In relation to the constitutional provisions, the claimants at ﬁrst instance referred to the regional Constitution of Lower Austria, which stipulates climate protection as an objective of provincial governance. However, the Constitutional Court denied its application.101 At the federal level, according to the Constitutional Court, the Constitutional Act on Sustainability may only guide the interpretation of provisions which already have the aim of environmental protection. It cannot add environmental protection as an interpretative principle to other acts. The examples illustrate that the enforcement of the Paris Agreement takes place within national law and depends on its interpretation. This also holds true for the interpretation of other international obligations. Once the Paris Agreement’s climate goals apply at the domestic level, the courts deduce their meaning from the state’s NDC. For example, the High Court referred to the peak, plateau, and decline trajectory provided in the South African NDC and thus allowed for consideration of the country’s development needs. In the Thabametsi Case, the court’s afﬁrmation of the duty to consider climate change impacts left room for the authorities to weigh up development needs against climate protection.102 Accordingly, the Minister’s decision issued after the judgment granted the construction permit for the power plant. In Austria, the Federal 100 101 102 Vienna Airport Case (VfGH), n. 17 above, para. 204, p. 62. B. Hollaus, ‘Austrian Constitutional Court: Considering Climate Change as a Public Interest is Arbitrary – Refusal of Third Runway Permit Annulled’ (2017) 11(3) Vienna Journal on International Constitutional Law, pp. 467–77, at 472. Thabametsi Case, n. 16 above, para. 35, p. 14. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 51 Administrative Court deduced from the EU’s NDC that the aviation sector should reduce its emissions, because otherwise Austria would fail to contribute to the EU’s 40% mitigation target.103 This interpretation led the court to give special weight to climate protection as one of the ‘other public interests’ interpreted in light of international climate change law. The Constitutional Court instead followed the apellants’ argument that ‘the environmental impacts are to be assessed against domestic environmental degradation, not against the global climate’.104 The courts’ functions of law creation and law enforcement depend not only on what the courts do or how they reason. Their hybrid function also depends highly on what national procedural law allows them to do. The Austrian Federal Administrative Court (at ﬁrst instance) fully reviewed and replaced the administrative decision. In contrast, the South African High Court referred the decision to the Minister. According to section 2(d) and (e)(iii) of the Promotion of Administrative Justice Act (PAJA), the courts review the administrative conduct in its entirety if it is ‘materially inﬂuenced by an error of law’. However, it is not in the power of the courts to substitute the administrative decision.105 The judgment of the Austrian Constitutional Court, in turn, highlights the restricted scope of review, characteristic of its role as a constitutional court. It did not delve into the ‘ﬁne-tunings’106 of the climate change assessment but focused exclusively on constitutional norms. The role of the courts within their domestic legal systems determines their role in the international legal order. Considering the importance of domestic contexts, the following question arises: how can the international legal system make sense of domestic jurisprudence relating to the international climate change regime? The following section advocates a comparative approach to domestic court decisions considering their embeddedness in national contexts. 5.         It has been shown that legal scholarship on the role of domestic courts in the international climate change regime mainly follows a governance approach. It therefore risks falling short of assessing the courts’ double-sided function of law creation and law enforcement as well as discussing the methodological preconditions necessary for understanding this function. The scholarly debate on the role of domestic courts in international law also provides valuable insights into the role of courts in international climate change law. It differentiates their functions and develops the methodological tools to assess them. As illustrated by the case studies, it provokes a range of questions. Is it still possible to frame domestic courts as enforcers and creators of international law once the national legislature has established the state’s contributions to the 103 104 105 106 Vienna Airport Case (BVwG), n. 17 above, pp. 125–6. Vienna Airport Case (VfGH), n. 17 above, para. 30, p. 12. Thabametsi Case, n. 16 above, p. 45, para. 116. Kirchengast et al., n. 91 above, p. 257. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 52 Transnational Environmental Law, 9:1 (2020), pp. 37–54 Paris Agreement’s climate goals? Can we still think of the courts as ‘translators of international law’107 considering that international law serves only a goal-setting function combined with procedural obligations? One possible pathway suggested by Roberts is to approach domestic climate change cases with a comparative method. It helps to explain how different actors interpret and apply international legal norms.108 The comparison carves out similarities and differences in the process of translation of international law into (sub)national legal orders. These similarities and differences are even more important with regard to the differentiated approach taken by the Paris Agreement. The international climate change regime itself is built on different obligations for different parties. Certainly, the obligations of conduct apply to all parties. However, the states bear common but differentiated responsibilities and NDCs differ from developing to developed countries.109 While differentiation under the Paris Agreement is subtle, it is clear from its Article 4(4) that developed countries ‘should continue taking the lead’.110 Geographical and economic preconditions, therefore, are anchored at the international level. As Article 4(2) of the Paris Agreement connects the obligation of conduct – namely ‘to pursue domestic mitigation measures’ – with the objectives of the NDCs, the substantive meaning of this obligation depends on domestic contexts. The legal architecture asks scholarship to take account of international law and domestic contexts at the same time. Following Roberts’ suggestion to compare the role of domestic courts, the aim and result of the comparison are as follows. Rather than seeking to ﬁx the ‘actual and proper’111 role of domestic courts, the comparison explores the contexts that allow domestic courts to link international obligations of conduct with national obligations of result. In this way international legal scholarship can make sense of domestic judicial decisions in the climate change regime and, at the same time, acknowledge the importance of domestic contexts. The purpose of the comparison, in turn, sheds light on the ‘object of juxtaposition, the tertium comparationis’.112 The obligations of conduct originating from international law serve as a common frame for comparative analyses. Each party to the Paris Agreement is subject to the duty to prepare, communicate, and maintain its NDC. The comparative perspective assesses the ability of domestic courts to ﬁll this process with substantive requirements. Such an undertaking ought to consider the constitutional and administrative provisions at the national level. The indeterminate legal terms in domestic administrative law, as well as the role of environmental provisions in 107 108 109 110 111 112 See Knop, n. 31 above. Roberts et al., n. 13 above, p. 7. Rajamani, n. 10 above, p. 501. Arts 3 and 4(4) Paris Agreement. Roberts, n. 6 above, p. 59. M. Mehling, ‘The Comparative Law of Climate Change: A Research Agenda’ (2015) 24(3) Review of European, Comparative & International Environmental Law, pp. 341–52, at 348. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press Anna-Julia Saiger 53 national constitutions, shape the margins open to the courts.113 Procedural law and the relevance of subjective rights to access the courts need also to be considered. Additionally, legal culture and institutional backgrounds play an important role as they constitute the contexts of jurisprudence.114 The comparative analysis needs to question its own scholarly standpoint. This relates to a discussion of the selected cases and the sources that are used to assess and compare the role of domestic courts in international law.115 A context-sensitive comparison also integrates the courts’ motivations and selfconceptions. It may reveal that domestic courts use (weak) international obligations for their own purposes to counterbalance the inﬂuence of other courts or branches of government. In this sense, the role of courts is central to the South African legal system. This relates to the current political situation. Because of corruption issues in the executive and legislative branches, increased trust is placed on the judiciary.116 The Austrian case illustrates this point differently. The Federal Administrative Court was established in 2014 as part of a major reform of administrative appeal procedures. Previously, the environmental senate was the competent appeal body in the Vienna Airport Case.117 The reform brought in judicial review of administrative provisions for the ﬁrst time. Much criticism was directed at the three-year-old Federal Administrative Court for its decision, and its competence to substitute administrative acts was challenged.118 In a reaction to this judgment, the Constitutional Court rather surprisingly took over the case instead of denying competence, which would have seen the case referred to the Higher Administrative Court. Moreover, it delivered its judgment within a very short time frame.119 The decision of the Constitutional Court as well as that of the Federal Administrative Court cannot be read without considering the institutional contexts and hierarchy between the two institutions.120 The events following the Vienna Airport Case highlight the national consciousness of the court’s function of creating and enforcing international climate change law by linking international obligations with national law. In the aftermath of the judicial decisions, the National Council (the Lower Chamber of the Federal Parliament) voted for a constitutional change which would integrate the constitutional objectives of economic growth, employment and a competitive economic location into the Constitutional Act on Sustainability.121 Such changes hamper the court’s ability to refer to climate protection 113 114 115 116 117 118 119 120 121 See Sections 4.1. and 4.2 above. In South Africa, where common law inﬂuences merge with Dutch-Roman legal traditions, the Constitution plays a pivotal role: F. du Bois, Wille’s Principles of South African Law, 9th edn (Juta, 2007); U. Kischel, Rechtsvergleichung (C.H. Beck, 2015), pp. 682–3. Mehling, n. 112 above, p. 351. Roberts, n. 6 above, p. 88. H.V. Vuuren, ‘South Africa: Democracy, Corruption and Conﬂict Management’, Centre for Development and Enterprise & Legatum Institute, ‘Democracy Works’ Conference Paper, Apr. 2013, pp. 14, 15, available at: https://www.cde.org.za. Hollaus, n. 101 above, p. 468. Kirchengast et al., n. 84 above, p. 130. Kirchengast et al., n. 91 above, p. 257. For a discussion of institutional contexts see R. Mann, ‘Non-ideal Theory of Constitutional Adjudication’ (2018) 7(1) Global Constitutionalism, pp. 14–53. Amendment to the Constitutional Act on Sustainability, IA 2172/A (XXV GP), 17 May 2017. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press 54 Transnational Environmental Law, 9:1 (2020), pp. 37–54 and to link the international obligations of conduct to national obligations of result. International legal scholarship should remain attentive towards these changes and understand national contexts from an international law perspective. 6.  This article stresses the importance of comparative approaches towards understanding the role of domestic courts in the international climate change regime since the Paris Agreement. The interconnectedness of international obligations of conduct and domestic substantive provisions, as well as domestic institutional and extra-legal contexts, challenges international legal scholarship to consider both levels of lawmaking. Against this background, climate change litigation may become an opportunity to (re)discuss the role of domestic courts in the international legal architecture. The proposed context-sensitive comparison allows for an understanding of the role of domestic courts within the international climate change regime while, at the same time, paying tribute to the bottom-up regulatory approach of the Paris Agreement. Rather than looking at the ‘yes or no’ question of whether domestic courts act in favour of climate protection, it embraces the nuanced conditions for the integration of international law into national legal systems. https://doi.org/10.1017/S2047102519000256 Published online by Cambridge University Press
https://openalex.org/W3133965623	https://www.researchsquare.com/article/rs-107432/latest.pdf	English	null	Empowering large chemical knowledge bases for exposomics: PubChemLite meets MetFrag	Journal of cheminformatics	2,021	cc-by	14,326	Todor Kondic University of Luxembourg Luxembourg Centre for Systems Biomedicine: Universite du Luxembourg Luxembourg Centre for Systems Biomedicine https://orcid.org/0000-0001-6662-4375 ff Paul Thiessen NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-1992-2086 Jian Zhang NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-6192-4632 Evan Bolton NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-5959-6190 Emma Louise Schymanski (  emma.schymanski@uni.lu ) Emma Louise Schymanski (  emma.schymanski@uni.lu ) LCSB, University of Luxembourg https://orcid.org/0000-0001-6868-8145 Empowering Large Chemical Knowledge Bases for Exposomics: Pubchemlite Meets Metfrag Emma Louise Schymanski (  emma.schymanski@uni.lu ) LCSB, University of Luxembourg https://orcid.org/0000-0001-6868-8145 Todor Kondic University of Luxembourg Luxembourg Centre for Systems Biomedicine: Universite du Luxembourg Luxem Centre for Systems Biomedicine https://orcid.org/0000-0001-6662-4375 Steffen Neumann Leibniz Institute of Plant Biochemistry: Leibniz-Institut fur Pflanzenbiochemie https://orcid.org/0000-000 7192 Paul Thiessen NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-1992-2086 Jian Zhang NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-6192-4632 Evan Bolton NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-5959-6190 Empowering Large Chemical Knowledge Bases for Exposomics: Pubchemlite Meets Metfrag Emma Louise Schymanski (  emma.schymanski@uni.lu ) LCSB, University of Luxembourg https://orcid.org/0000-0001-6868-8145 Todor Kondic University of Luxembourg Luxembourg Centre for Systems Biomedicine: Universite du Luxembourg Luxem Centre for Systems Biomedicine https://orcid.org/0000-0001-6662-4375 Steffen Neumann Leibniz Institute of Plant Biochemistry: Leibniz-Institut fur Pflanzenbiochemie https://orcid.org/0000-000 7192 Paul Thiessen NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-1992-2086 Jian Zhang NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-6192-4632 Evan Bolton NCBI: National Center for Biotechnology Information https://orcid.org/0000-0002-5959-6190 Abstract Compound (or chemical) databases are an invaluable resource for many scientific disciplines. Exposomics researchers need to find and identify relevant chemicals that cover the entirety of potential (chemical and other) exposures over entire lifetimes. This daunting task, with over 100 million chemicals in the largest chemical databases, coupled with broadly acknowledged knowledge gaps in these resources, leaves researchers faced with too much – yet not enough – information at the same time to perform comprehensive exposomics research. Compound (or chemical) databases are an invaluable resource for many scientific disciplines. Exposomics researchers need to find and identify relevant chemicals that cover the entirety of potential (chemical and other) exposures over entire lifetimes. This daunting task, with over 100 million chemicals in the largest chemical databases, coupled with broadly acknowledged knowledge gaps in these resources, leaves researchers faced with too much – yet not enough – information at the same time to perform comprehensive exposomics research. Furthermore, the improvements in analytical technologies and computational mass spectrometry workflows coupled with the rapid growth in databases and increasing demand for high throughput “big data” services from the research community present significant challenges for both data hosts and workflow developers. This article explores how to reduce candidate search spaces in non-target small molecule identification workflows, while increasing content usability in the context of environmental and exposomics analyses, so as to profit from the increasing size and information content of large compound databases, while increasing efficiency at the same time. In this article, these methods are explored using PubChem, the NORMAN Network Suspect List Exchange and the in silico fragmentation approach MetFrag. A subset of the PubChem database relevant for exposomics, PubChemLite, is presented as a database resource that can be (and has been) integrated into current workflows for high resolution mass spectrometry. Benchmarking datasets from earlier publications are used to show how experimental knowledge and existing datasets can be used to detect and fill gaps in compound databases to progressively improve large resources such as PubChem, and topic-specific subsets such as PubChemLite. PubChemLite is a living collection, updating as annotation content in PubChem is updated, and exported to allow direct integration into existing workflows such as MetFrag. The source code and files necessary to recreate or adjust this are jointly hosted between the research parties (see data availability statement). Research article DOI: https://doi.org/10.21203/rs.3.rs-107432/v1 Version of Record: A version of this preprint was published on March 8th, 2021. See the published version at https://doi.org/10.1186/s13321-021-00489-0. Page 1/25 Abstract This effort shows that enhancing the FAIRness (Findability, Accessibility, Interoperability and Reusability) of open resources can mutually enhance several resources for whole community benefit. The authors explicitly welcome additional community input on ideas for future developments. Introduction Compound (or chemical) databases are an invaluable resource for many scientific disciplines. Through the joint evolution over the last decade of high resolution mass spectrometry (HR-MS), cheminformatics techniques and openly available compound databases, a whole new world for identifying small molecules in complex samples has emerged. Despite many advances, chemical identification is still generally considered a bottleneck in many research fields (see e.g. [1, 2]). Interest in the exposome [3] and the related exposomics field has increased as awareness of the influence of the external environment on health and disease has increased [4]. Exposomics requires researchers to find and identify relevant chemicals that cover the entirety of potential (chemical and other) exposures over entire lifetimes [4–6], significantly adding to the identification challenge. Scientific disciplines such as environmental science, metabolomics, forensics and exposomics are focusing increasingly on high throughput data exploration with high resolution mass spectrometry (HR-MS) techniques [4, 7, 8]. Mass spectral libraries, which can be used to obtain rapid tentative identifications of relatively high confidence [9–11] still only cover a fraction of chemical information resources relevant in exposomics [9], metabolomics [12] or in complex samples in general [13, 14]. This is especially true for HR-MS techniques, which are inherently limited by the availability of reference standards as well as the relative youth and lack of standardization in the field [9]. Alternative methods to annotate detected exact masses in HR-MS studies beyond spectral library searching began emerging around 2010 by searching compound (i.e., chemical) databases for possible candidates using the exact mass or calculated molecular formula, and ranking these using in silico techniques to sort candidates using the Alternative methods to annotate detected exact masses in HR-MS studies beyond spectral library searching began emerging around 2010 by searching compound (i.e., chemical) databases for possible candidates using the exact mass or calculated molecular formula, and ranking these using in silico techniques to sort candidates using the Page 2/25 Page 2/25 measured fragmentation information. The plethora of identification methods now available are described and compared in detail elsewhere [14–17]. A wide variety of (generally open) compound databases are typically used as information sources for these identification efforts, containing anything between tens to hundreds of thousands (e.g. KEGG [18], HMDB [19, 20], CompTox [21]) and tens of millions of structures (e.g. ChemSpider [22] and PubChem [23, 24]). Introduction Most of these resources and, consequently, the number of candidates per exact mass/formula, are expanding significantly over time. Typical queries with smaller databases return tens to hundreds of candidates, whereas typical queries with large databases such as PubChem now return thousands to tens of thousands of candidates per exact mass/formula query. For instance, querying HMDB, CompTox and PubChem with the formula C10H14N2 via the MetFrag [25, 26] web interface (12 August 2020) returns 4, 225 and 3,704 candidates, respectively. A major challenge in correctly identifying a chemical based on exact mass (or formula) and fragmentation information alone arises due to the relatively little information conveyed in the fragmentation spectrum. During one open community evaluation approach, the 2016 Critical Assessment of Small Molecule Identification (CASMI) contest, participants were provided 208 challenges with fragmentation information and candidate query sets retrieved from ChemSpider [16]. Using fragmentation information alone, participants were able to rank between 24 (11.5%) and 70 (33.7%) of these 208 challenges correctly in first place [16]. However, combining this fragmentation information with other forms of information (e.g. references, retention time information) yielded up to 164 (78.8%) challenges correctly ranked in first place when combining all participant methods over the same ChemSpider candidate sets [16]. Separately, a detailed evaluation of MetFrag combining retention time information with various scoring terms available via ChemSpider (5 different literature terms) and PubChem (PubMed Count and Patent Count) for 473 environmentally relevant standards was performed. This revealed that ranking results were improved from 22 to 89% with ChemSpider and from 6 to 71% with PubChem (with 34 and 71 million entries respectively at the time) [25]. In summary over these evaluations and more; better ranking performance is achieved with small, select databases, at the risk of missing the correct answer [27], while the use of additional metadata (expert knowledge, additional context) is necessary to improve the results for practical use, especially when using very large compound databases to search for candidates. Another challenge, especially for exposomics, is database choice. Being a mix between metabolomics and environmental concepts and challenges, exposomics methods need, on the one hand, the biological context of pathway and metabolomics resources (generally small, specialist metabolite databases such as HMDB and KEGG), versus the wide coverage required to capture “chemical space” which, in environmental contexts, generally means PubChem or ChemSpider. Introduction Although recent works mention the need for an “exposomics database”, much of the necessary knowledge is already in the public domain to some extent, but under rapid development and scattered over an ever-growing number of resources. Notable recent developments include the CompTox Chemicals Dashboard, covering 882,000 (August 2020) environmentally and toxicologically-relevant compounds [21] and the Blood Exposome Database [28], which, although specifically designed for the blood matrix, still contains over 64,000 compounds. Large compound databases such as PubChem have content in common with many of the openly available smaller databases, but at a size of 111 million compounds (October 2020), PubChem also contains many (tens of) millions of entries that are not relevant to the exposomics context. Beyond the database choice, common criticisms of small molecule identification coupled to compound databases arising from users over the years include the fact that newly-discovered and/or relevant compounds such as emerging chemicals, transformation products and metabolites are missing from, or hard to add to, these databases for a typical researcher. If these compounds are present, these tend to have very low metadata scores and thus common environmental knowledge of transformations or emerging chemicals cannot often be found effectively Page 3/25 Page 3/25 during identification efforts. As a result (and also to increase efficiency), many groups in the environmental community have taken to compiling their own lists of relevant chemicals (commonly termed “suspect lists” within this community [7]). The NORMAN Suspect List Exchange (NORMAN-SLE) [29] is one initiative that arose to address NORMAN Network [30, 31] member needs to exchange this information as a result of a collaborative trial in 2014 [32], and to date is host to over 73 specialised NORMAN member contributed lists of chemicals of interest. during identification efforts. As a result (and also to increase efficiency), many groups in the environmental community have taken to compiling their own lists of relevant chemicals (commonly termed “suspect lists” within this community [7]). The NORMAN Suspect List Exchange (NORMAN-SLE) [29] is one initiative that arose to address NORMAN Network [30, 31] member needs to exchange this information as a result of a collaborative trial in 2014 [32], and to date is host to over 73 specialised NORMAN member contributed lists of chemicals of interest. Introduction With a view on this “current state”, this article investigates how very large compound databases, or knowledge bases, such as PubChem, could be empowered to support HR-MS-based small molecule identification efforts in the context of exposomics. This article describes initial collaborative efforts on how to improve the performance of the PubChem integration into the in silico identification approach MetFrag. Since the first release of MetFrag in 2010, PubChem has grown from 25 million to now 111 million compounds, with an accompanying steadily worsening rank performance and increasing strain on resources due to the rapidly increasing candidate numbers. Three main aspects of these collaborative discussions are presented in this article: (1) the creation of a small, exposomics- relevant subset of PubChem – named PubChemLite – for efficient candidate queries, which has already been integrated into existing HR-MS workflows and teaching efforts; (2) progressive integration of environmentally- relevant expert knowledge to mitigate identified knowledge gaps in PubChem annotation content, based on analysis of previous benchmarking sets and the NORMAN-SLE content; and (3) how annotation content can be leveraged for easier interpretation of results. As a result, this article focuses heavily on PubChem, MetFrag and the NORMAN-SLE, with the view that the ideas presented here could be extended to other knowledge bases and other in silico identification approaches based on HR-MS. Creating “PubChemLite” for Exposomics An RMarkdown file to visualize the content (categories and subcategories) of PubChemLite as an interactive sunburst plot (for a static version see Fig. 2) using the 14 January 2020 tier1 version is included as Additional File 1 and is also available on the ECI GitLab pages [36, 37]; further details are in the Methods section below. A benchmark dataset of 977 de-duplicated compounds (see Additional File 2) was created by merging chemicals from previous evaluations [16, 25] (predominantly environmentally relevant) as described in the Methods. MetFrag was run with different versions of PubChemLite as well as CompTox (7 March 2019 release [38]) using comparable scoring terms. A summary of the results shown in Fig. 3 includes calculations both without (green) and with (blue) the use of MS/MS information (in silico fragmentation score and MS library matching scores). Further parameter details are given in the Methods section, with tables included in Additional File 3. Overall, CompTox and PubChemLite perform comparably; initially CompTox had fewer missing entries (grey shading) due to their earlier concerted efforts to add compounds of environmental interest, including transformation products (these gaps may well be smaller with the new data release). These gaps were closed progressively in PubChemLite as described in the next section “Identifying and Filling Gaps in PubChem Annotation Content”. Furthermore, early results (see Additional File 3 Figures S1 and S2, Tables S1 and S2) showed that both versions of PubChemLite, tier0 and tier1, performed almost identically even on environmental substances of interest, such that finally, one “PubChemLite” for exposomics will be created, equivalent to tier1 plus the two additional categories as shown in Fig. 1 [39]. Results from this version are also shown in Fig. 3. The results in Fig. 3 show that, while annotation information alone leads to good ranking performance (~ 70–73% ranked first, dark green shaded results), the MS/MS information is essential for further improvements (~ 79–83% ranked first, dark blue shaded results). This is discussed further below. The PubChemLite results on the two initial versions (20 November 2019 and 14 January 2020) also clearly show that ~ 8% of the benchmark dataset were missing from PubChemLite. A detailed interrogation of the benchmark set of 977 reference standards from Eawag and UFZ revealed that – as commented by the community over many years – detailed annotation information was missing for well-known relevant transformation products in PubChem. Creating “PubChemLite” for Exposomics Creating “PubChemLite” for Exposomics Since a very large proportion of the PubChem database (> 60%) is sourced from purchasable screening libraries from chemical vendors, where the chemicals are generally produced in relatively small amounts (e.g. mg) in a laboratory setting, the vast majority of these chemicals are highly unlikely to be detectable in either the environment or biological samples. Thus, instead of the current status quo, i.e. searching the entire PubChem database and using metadata scores to “up-prioritize” interesting candidates (i.e., processing tens of thousands of candidates per mass, to only obtain tens to hundreds of interesting entries), the first step investigated the creation of relevant subsets of PubChem for more efficient queries. This was done by selecting relevant sections of the “PubChem Compound Table of Contents” (PubChem Compound TOC) Classification [33] as shown in Fig. 1. Further details are given in the Methods section. Page 4/25 Initially, two versions of PubChemLite were created. The environmental selection (PubChemLite tier0), formed of the yellow-shaded categories in Fig. 1, shortened to “AgroChemInfo, DrugMedicInfo, FoodRelated, PharmacoInfo, SafetyInfo, ToxicityInfo, KnownUse”, whereas the exposomics selection (PubChemLite tier1) had the additional purple-shaded category, shortened to “BioPathway”, which contained the additional biological information categories relevant to metabolomics and exposomics. Entries were merged by InChIKey first block (the structural skeleton), and total Patent Counts and Literature Counts were calculated over the merged entries (full details in the Methods section). Each category was added as an additional column, where each entry was assigned a value that was a (merged) count of the sub-categories, and a total annotation count column was also added, summing the presence in top categories only (for further details, see methods). Initial versions (20 November 2019 [34] / 14 January 2020 [35]) contained 315,843 / 316,810 entries in tier0 (environmental collection) and 361,976 / 363,911 entries in tier1 (exposomics). In other words, the 103 M entries of PubChem (at the time) were collapsed down to two datasets of approximately 316 K and 360 K compounds. An RMarkdown file to visualize the content (categories and subcategories) of PubChemLite as an interactive sunburst plot (for a static version see Fig. 2) using the 14 January 2020 tier1 version is included as Additional File 1 and is also available on the ECI GitLab pages [36, 37]; further details are in the Methods section below. approximately 316 K and 360 K compounds. Creating “PubChemLite” for Exposomics This accounted for 37 of the 57 missing entries in the January 14, 2020 tier0 version and is discussed further in the next section. Identifying and Filling Gaps in PubChem Annotation Content During previous evaluations of MetFrag specifically [25], and in silico identification approaches for HR-MS in general during e.g. CASMI [16], the focus has generally been on evaluating the methods themselves, aiming for objective evaluation. The use of identification approaches in typical real-life scenarios, however, often requires additional subjectiveness to provide interpretation, not just identification. Thus, the material in this article should not be viewed as an evaluation of MetFrag itself (which has not changed), but rather demonstrates how improving the underlying database and associated functionality can help to improve outcomes for users (i.e. the ability to find relevant chemicals) in the context of exposomics. In other words, this has been an opportunity to investigate and improve the annotation content (i.e. information content beyond structural properties) in PubChem for exposomics. As Fig. 3 reveals, 57 chemicals from the benchmark set were missing in the early versions of PubChemLite, many of which were well-known transformation products in environmental studies. Since adding annotation content requires also sufficient provenance and evidence to support the annotation, the NORMAN-SLE [29, 43], which now has its own Classification Browser [44] in PubChem (see Fig. 4) was browsed for suitable suspect lists containing annotation content. Initial efforts concentrated on list S60 (SWISSPEST19) [45], a list of pesticides and transformation products / metabolites documented by Kiefer et al. [46]. This list contained parent-transformation product mappings, plus the Page 5/25 link to information about agrochemical use (since the focus was on pesticides). The list was modified into a “predecessor / successor” mapping form (to avoid terminology clashes within other sections of PubChem) and added, with full provenance, into a new “Transformations” section in the individual PubChem records (see Fig. 5). Accompanying statements on “Agrochemical Transformations” within the agrochemical sections were also added, for example “Folpet has known environmental transformation products that include Phthalimide, Phthalamic acid, and Phthalic acid” [47]. The PubChemLite version created 22 May 2020 [48] included these new annotations, with fewer missing entries and slightly better ranks (see Fig. 3). Since this only focused on the agrochemicals (pesticides), the many pharmaceutical (and other) transformation products among the Eawag dataset were still missing. Creating “PubChemLite” for Exposomics While these are all present in MassBank [49] (S1 in the NORMAN-SLE [50]), this dataset does not come with appropriate annotation content or provenance. Instead, the Supporting Information from Schollee et al. [51] provided suitable parent-TP mappings to create the predecessor-successor tables, which was merged with the Eawag classification information (with permission and support from Juliane Hollender) and added as list S66 [52]. This collection, together with list S68 HSDBTPS [53], resulted in the greater coverage in the June 2020 [48] and October 2020 [39] versions (see Fig. 3), with only 16 missing entries (15 in October) remaining. These remaining 16 entries could not be clearly related to any specific NORMAN-SLE lists to add further annotation content at this stage; although annotation content is being progressively added in separate efforts – as is evident from the one less missing entry in October. link to information about agrochemical use (since the focus was on pesticides). The list was modified into a “predecessor / successor” mapping form (to avoid terminology clashes within other sections of PubChem) and added, with full provenance, into a new “Transformations” section in the individual PubChem records (see Fig. 5). Accompanying statements on “Agrochemical Transformations” within the agrochemical sections were also added, for example “Folpet has known environmental transformation products that include Phthalimide, Phthalamic acid, and Phthalic acid” [47]. The PubChemLite version created 22 May 2020 [48] included these new annotations, with fewer missing entries and slightly better ranks (see Fig. 3). Since this only focused on the agrochemicals Leveraging Annotation Content in Exposomics To address this, the benchmarking dataset (n = 977) used above (with MS/MS information available) was subset according to the availability of information in the Agrochemical Information category (creating a subset of n = 318), and evaluated with scoring terms relevant to the annotation type, as shown in the blue entry in Fig. 6. This mimics, to a certain extent, a typical suspect screening workflow where the main interest is in finding and confirming pesticides in an environmental sample. As shown, adding MS/MS information (MetFrag in silico fragmentation plus MoNA similarity score) increased the correctly ranked chemicals in first place to 90.6% for those agrochemicals that were also in the benchmarking set. If the database (in this case PubChemLite tier0 12 Jun 2020 version) had been restricted to agrochemicals only this would have risen to 94.3%, as some non-agrochemical available) was subset according to the availability of information in the Agrochemical Information category (creating a subset of n = 318), and evaluated with scoring terms relevant to the annotation type, as shown in the blue entry in Fig. 6. This mimics, to a certain extent, a typical suspect screening workflow where the main interest is in finding and confirming pesticides in an environmental sample. As shown, adding MS/MS information (MetFrag in silico fragmentation plus MoNA similarity score) increased the correctly ranked chemicals in first place to 90.6% for those agrochemicals that were also in the benchmarking set. If the database (in this case PubChemLite tier0 12 Jun 2020 version) had been restricted to agrochemicals only this would have risen to 94.3%, as some non-agrochemical available) was subset according to the availability of information in the Agrochemical Information category (creating a subset of n = 318), and evaluated with scoring terms relevant to the annotation type, as shown in the blue entry in Fig. 6. This mimics, to a certain extent, a typical suspect screening workflow where the main interest is in finding and confirming pesticides in an environmental sample. As shown, adding MS/MS information (MetFrag in silico fragmentation plus MoNA similarity score) increased the correctly ranked chemicals in first place to 90.6% for those agrochemicals that were also in the benchmarking set. Leveraging Annotation Content in Exposomics If the database (in this case PubChemLite tier0 12 Jun 2020 version) had been restricted to agrochemicals only this would have risen to 94.3%, as some non-agrochemical isomers still outscored several entries based on the literature and patent values. The performance would not be able to rise much higher than 94% with this dataset, however, since there are multiple agrochemical isomers present in the dataset where the less-well-known (but often structurally related) isomers ranked lower because of less supporting metadata. For instance, for secbutylazine (CID 23712), the candidate terbutylazine, CID 22206 was ranked first and secbutylazine, CID 23712 was third, while another isomer propazine CID 4937 was second. All three isomers were in the dataset. In this case, both the in silico fragmenter and MoNA similarity scores captured these three isomers in the correct order (secbutylazine first, terbutylazine second, propazine third), showing that the experimental evidence is still crucial in distinguishing isomers - or indicating whether they are indistinguishable on given evidence. Terbutylazine was correctly ranked first for its corresponding entry (see Table 1). ked first for its corresponding entry (see Table 1). Table 1 Candidate score distributions for three isomers/isobars of formula C9H16ClN5 in the agrochemical dataset. Values for the correct candidate in each case are bolded. Only the scores for the top 5 candidates (of 37) are shown. Name (CID) Terbutylazine (22206) Propazine (4937) Secbutylazine (23712) MetFrag Scores 4.96; 3.45; 2.77; 1.93; 1.59 4.46; 3.88; 2.27; 1.81; 1.58 4.96; 3.52; 2.78; 1.92; 1.57 Fragmenter Score 351; 250; 351; 239; 126 247; 295; 251; 170; 106 398; 303; 403; 272; 135 MoNA Similarity 0.959; 0.672; 0.987; 0.0; 0.0 0.638; 0.841; 0.661; 0.0; 0.0 0.971; 0.703; 0.998; 0.0; 0.0 PubMed Count 282; 127; 0; 11; 1 282; 127; 0; 11; 1 282; 127; 0; 11; 1 Patent Count 10935; 8900; 1990; 6636; 6861 10935; 8900; 1990; 6636; 6861 10935; 8900; 1990; 6636; 6861 Annotation Count 5; 5; 4; 4; 5 5; 5; 4; 4; 5 5; 5; 4; 4; 5 AgroChemInfo 5; 4; 3; 3; 3 5; 4; 3; 3; 3 5; 4; 3; 3; 3 Rank 1 of 37 2 of 37 3 of 37 Table 1 Using this benchmarking dataset alone, taking PubChemLite and using the specific topic information for agrochemicals, most candidates were ranked 1st and the worst rank for a chemical was 3rd. Leveraging Annotation Content in Exposomics The results presented in Fig. 3 detailed the use of rather generic metadata terms (literature counts, patent counts, total annotation counts). However, one aim of setting up PubChemLite was not only to merge several “useful” categories for exposomics, but to leverage the information within these categories (providing interpretation about candidates in candidates sets). The smallest annotation category in PubChemLite, the agrochemicals, was taken as an additional benchmarking dataset (1336 chemicals, 22 Jan 2020, see Additional File 4) to investigate the influence of database size and the additional scoring terms on the ranking results. Since this was to mimic an environmental investigation interested in detecting agrochemicals (i.e. a “suspect screening” approach [7]), the “agrochemical score”, i.e. how many agrochemical categories exist in PubChem for that chemical, was used as an additional scoring term in MetFrag (details in the Methods). The results are shown as the green entries in Fig. 6; the exact numbers are given in Additional File 3 (Table S3). With a full PubChem query and using only literature and patent information to score, only 58% of entries were correctly ranked in first place (which is not unexpected, as e.g. pharmaceuticals, industrial chemicals or even metabolites with the same mass may have larger literature or patent counts). When the database was restricted to the candidates in PubChemLite using the same scoring terms (literature and patent counts), this increased to 70%. However, adding the Agrochemical Score improved this further to 79.2%, demonstrating the potential usefulness of individual category-based scoring terms to help select relevant chemicals for further verification. In terms of computational efficiency, the last 101 queries (entries 1236–1336) of the Agrochemicals query took 11 minutes to complete with PubChemLite tier1 (query run 21 Jan 2020), while the equivalent query with the full PubChem database and scoring terms took 164 minutes (query run 26 Jan 2020). This results in approx. 6.5 sec per query for PubChemLite, versus 97 seconds per query for a full PubChem query (note: both queries were without fragmentation). Page 6/25 Since this is purely annotation-based scoring, it is imperative to use additional experimental information such as fragmentation information and further verification with reference standards before any claims of higher confidence annotation are made [11]. Conclusions The need to cover the “entire chemical space” in exposomics research is a huge challenge for researchers and database resources alike (and currently unachievable – due to our inability to define chemical space completely). This article explores the use of annotation content of very large compound databases, i.e. compound knowledge bases, to create meaningful and efficient subsets relevant to specific use cases, specifically aimed at creating subsets of PubChem most relevant for exposomics. The resulting PubChemLite is a dynamic yet efficient database that grows as the respective (and relevant) annotation categories grow in PubChem, and is built and deposited regularly to allow integration with existing HR-MS identification approaches such as MetFrag [26, 58] and comprehensive MS workflows such as patRoon [59]. The subcategories present in PubChemLite allow end users a certain a degree of individual or sample-wide interpretation of the results, such that broad chemical categories become obvious amongst suggested candidates. These can be used as scoring terms or hard filters, depending on user choice, and subsets of the database could serve as large suspect lists if desired. PubChemLite is already in use in several research projects. Feedback on the approach and further integration into other resources and workflows is greatly welcomed. Further developments are being made behind the scenes to streamline the ideas presented in this manuscript for the community in other ways. The code and all necessary files are available (see availability statement), such that expert users can build and compile their own subsets of PubChem using any of the categories available in the PubChem Table of Contents Classification Browser [33] by defining their own input “bit sets”. To address the “data gap” issue of highly-relevant compounds missing in existing compound databases (a broadly acknowledged weakness and argument frequently applied against using compound databases for HR-MS-based tentative identification efforts), this article also explores how knowledge gaps can be assessed and filled, as exemplified with environmentally-relevant information from the NORMAN Network. A coupled deposition and annotation workflow has been set-up between PubChem and the NORMAN-SLE, allowing the deposition of environmentally relevant substances into PubChem and the progressive integration of the accompanying (relevant) annotation content, with full traceability to the original data sources. The examples covered in detail here included transformation product and agrochemical use cases. Leveraging Annotation Content in Exposomics Creating a similar pharmaceutical subset (as opposed to agrochemicals) using the “DrugMedicInfo” category yielded similar results (most ranked first, worst rank of 3rd ) using either DrugMedicInfo or PharmacoInfo as scoring terms (see Additional File 3, Figure S3). For a more generic category such as ToxicityInfo, most were ranked 1st or 2nd, but the worst rank was 12, indicating that this term may be less selective (see Additional File 3, Figure S3). Using patent and literature information alone (over the entire benchmark set), the worst rank was 27th, with 11 entries missing entirely. Thus, even though this dataset is of limited size (977 entries), the results indicate that there is a good chance that the top candidate will be among the Top 3 using PubChemLite for highly specific categories such as (agrochemicals, pharmaceuticals). On the other hand, more candidates will often have to be considered for less specific categories or Page 7/25 Page 7/25 questions (e.g. Toxicity Information) or when only the generic scoring terms are used. In the context of practical use of HR-MS for answering real life questions, e.g. the presence of well-known chemicals in environmental or patient samples, considering only a few candidates (e.g. 1–3) versus hundreds or even thousands of candidates per mass is a great step forward for higher throughput interpretation of non-target screening results and coming to meaningful conclusions quicker. It is expected that greater granularity in the annotation information will improve the interpretability and applicability of this information in the future (for instance toxicity information is currently often only “information is present” and not “the substance is toxic”); efforts are being made to achieve this (beyond the scope of the current article). As a future perspective, the addition of extra information, such as partitioning information (e.g. logP, logKow or logD) and collision cross section (CCS) values, will also help in candidate selection in specific cases (although for isobars /isomers that are very similar, predictive values will often be very close). Efforts are currently underway to include XlogP3 [54] in future versions of PubChemLite to integrate within the retention time model already present in MetFrag [25]. Further, an initial version of PubChemLite (January 14, 2020 tier1) with CCS values contributed by CCSbase [55, 56] is also available on Zenodo [57] and in MetFrag web version [26] and is currently being evaluated in separate work. Conclusions Importantly, these integration efforts enhance both resources and help combine knowledge into a central location (thus increasing the FAIRness of the data) by reducing the Page 8/25 Page 8/25 isolation of the individual NORMAN-SLE lists while increasing the annotation (information) available in PubChem. The integration of content is occurring progressively with a focus on areas of high community interest and on those filling the largest gaps. Community input is very welcome to help focus these efforts to maximize the overall benefit. The content is available in a variety of formats across both resources for re-use. isolation of the individual NORMAN-SLE lists while increasing the annotation (information) available in PubChem. The integration of content is occurring progressively with a focus on areas of high community interest and on those filling the largest gaps. Community input is very welcome to help focus these efforts to maximize the overall benefit. The content is available in a variety of formats across both resources for re-use. While PubChemLite is an immediately accessible stepping-stone for HR-MS-based exposomics research, it is still only a small part of efforts towards a bigger picture solution for the exposomics challenge. Enhancing the annotation content of compound knowledge bases is clearly one way of improving the useability of very large knowledge bases. Dynamic and easy-to-use ways to subset and/or order the chemicals based on this annotation content (beyond creation of a MetFrag-specific output file) will be needed to improve the useability further. At some point, specialist users will need to be able to tell chemical knowledge bases what they want to find to improve their search results for their specific use case, rather than just taking the “best match” based on generic scores such as literature or annotation counts. Future efforts, beyond enhancing annotation content, will include continuing conversations with users and the community to develop functionality that can be applied either on the database side, or the workflow side, or both, to truly empower large compound knowledge bases for exposomics research and move from just identification towards more detailed interpretation of HR-MS datasets. Creating PubChemLite for MetFrag Creating PubChemLite for MetFrag Creating PubChemLite for MetFrag MetFrag currently has PubChem integrated via the RESTful API as well as a local mirror. Of the typically thousands of candidates that are retrieved using exact mass (with ppm error margin) or molecular formula queries, several candidates are returned that are eventually discarded (e.g. disconnected structures, which cannot be observed at the input mass or formula in the mass spectrometer, or other structures that cannot be processed by MetFrag). Since high resolution mass spectrometry rarely yields information on stereochemistry (there are exceptions for some substances e.g. when chiral chromatography is used), it is the default behaviour of MetFrag and many other approaches to merge candidates by the first block of the InChIKey (i.e. the structural skeleton) and present the users results displaying the stereoisomer with the highest score. For candidates merged by InChIKey first blocks, any ranking is usually driven by metadata rather than fragmentation, which does not usually contain sufficient information to distinguish stereoisomers, except for some tautomers. In MetFrag, this stereoisomer filtering can be switched on or off as desired. However, for larger (or complex) structures, the presence of stereoisomers can dramatically inflate candidate numbers and reduce calculation efficiency, often for little final gain. To create subsets of PubChem by annotation content category, firstly a Table of Contents fingerprint (TOC FP) was created for each of the PubChem Compound TOC entries (each bit representing presence or absence of information in that category for a compound) along with metadata indicating the relationship between the bits (e.g., subcategories of a given annotation). Then, mapping files containing the desired TOC entries were created. Finally the relevant data (compound information, patent and literature scores, plus the TOC fingerprints) was extracted by the compound identifier (CID) from the respective PubChem download files [60] using scripts that have been made available at the Environmental Cheminformatics group GitLab pages [61]. To create subsets of PubChem by annotation content category, firstly a Table of Contents fingerprint (TOC FP) was created for each of the PubChem Compound TOC entries (each bit representing presence or absence of information in that category for a compound) along with metadata indicating the relationship between the bits (e.g., subcategories of a given annotation). Then, mapping files containing the desired TOC entries were created. Creating PubChemLite for MetFrag For example, the InChIKey first block HXKKHQJGJAFBHI has 6 related CIDs in PubChemLite tier 0 (14 Jan 2020 version: 4, 111033, 439938, 446260, 7311736, 44150279), while 9 CIDs (4, 439938, 446260, 4631415, 7311735, 7311736, 16655457, 123598986, 140936702) match this InChIKey first block in the PubChem search interface (search date 22 May 2020 [62]). These rules were selected for maximum efficiency, resulting in the following behaviour that should be considered when interpreting the results. Firstly, collapsing all annotated CIDs by IKFB could result in the inclusion of different isotopic states and/or charges, which may not be included otherwise in MetFrag queries initiated by exact mass/formula and could otherwise prevent these candidates appearing in PubChemLite queries at their true exact mass/formula. In the context of efficient screening of masses for environmental, metabolomics or exposomics studies, matches with differing isotopic states are unlikely to be found in large amounts in these studies. In the cases that isotopically labelled standards are used, or isotopically labelled experiments are performed, other data interrogation techniques are usually necessary/recommended to capture these peaks in advance of identification efforts. For differing charge states, since these are usually accounted for in the upstream workflow by adjusting the adduct state, the current behaviour ensures a consistent “base state” for adjustment of charge in other parts of the workflow. Secondly, mixtures are currently discarded from PubChemLite files, as this would require an additional degree of manipulation (splitting and re-merging of the entries), which was not accounted for in the current version as this affects < 10K entries - of which a significant proportion are salts. It would be possible to address both issues in future versions should subsequent use cases deem this necessary. Finally, related CIDs are only included if that CID contains any annotation in at least one of the selected annotation categories. For example, the InChIKey first block HXKKHQJGJAFBHI has 6 related CIDs in PubChemLite tier 0 (14 Jan 2020 version: 4, 111033, 439938, 446260, 7311736, 44150279), while 9 CIDs (4, 439938, 446260, 4631415, 7311735, 7311736, 16655457, 123598986, 140936702) match this InChIKey first block in the PubChem search interface (search date 22 May 2020 [62]). As PubChem is changing daily, both in terms of numbers of chemicals and their annotation content, PubChemLite will not remain static. Creating PubChemLite for MetFrag Finally h l d ( d i f i d li l h TOC fi i ) d b To create subsets of PubChem by annotation content category, firstly a Table of Contents fingerprint (TOC FP) was created for each of the PubChem Compound TOC entries (each bit representing presence or absence of information in that category for a compound) along with metadata indicating the relationship between the bits (e.g., subcategories of a given annotation). Then, mapping files containing the desired TOC entries were created. Finally the relevant data (compound information, patent and literature scores, plus the TOC fingerprints) was extracted by the compound identifier (CID) from the respective PubChem download files [60] using scripts that have been made available at the Environmental Cheminformatics group GitLab pages [61]. Following this, and considering the current MetFrag behaviour, a set of rules was applied to the CIDs extracted from the TOC categories to generate a file that could be processed by MetFrag. Candidates that would be discarded later anyway (e.g. disconnected structures or other structures that cannot currently be processed by MetFrag) were Page 9/25 Page 9/25 Page 9/25 discarded up front. Further, CIDs were collapsed by the first block to have one “best matching” CID and mappings to all related CIDs. The rules applied were the following: discarded up front. Further, CIDs were collapsed by the first block to have one “best matching” CID and mappings to all related CIDs. The rules applied were the following: discarded up front. Further, CIDs were collapsed by the first block to have one “best matching” CID and mappings to all related CIDs. The rules applied were the following: 1. Retrieve all CIDs in PubChem with the desired annotation categories; 2. Map all CIDs to corresponding parent CIDs to obtain the neutral form, where available, imputing the annotation to the parent; 3. Collapse by InChIKey first block (IKFB), imputing total annotation to the IKFB, retaining the “best” CID (the most annotated CID for the given IKFB) and listing all related CIDs in a separate column, thus grouping all CIDs with annotation available; 4. Creating PubChemLite for MetFrag Remove all entries containing the following elements: Kr, Dy, Ir, La, Lu, Nd, Nb, Os, Pd, Pt, Pu, Pr, Re, Rh, Ru, Sm, Sc, Ag, Ta, Tc, Tb, Th, Tm, Ti, W, Ac, Am, Er, Eu, Gd, Hf, Ho, Xe, Yb, Rn, Sr, Be, Cm, Cf, Cs, Md, Pm, Fr, Pa, Np, Bk, Es, Fm, No, Lr, Rf, Db, Sg, Bh, Hs, Mt, Ds, Rg, Cn, Nh, Fl, Mc, Lv, Ts, Og; Fm, No, Lr, Rf, Db, Sg, Bh, Hs, Mt, Ds, Rg, Cn, Nh, Fl, Mc, Lv, Ts, Og; 5. Remove disconnected structures - as these will not be observed at the mass/formula of the query; 5. Remove disconnected structures - as these will not be observed at the mass/form 6. Remove charges from charged molecular formulae (but not the corresponding structures). These rules were selected for maximum efficiency, resulting in the following behaviour that should be considered when interpreting the results. Firstly, collapsing all annotated CIDs by IKFB could result in the inclusion of different isotopic states and/or charges, which may not be included otherwise in MetFrag queries initiated by exact mass/formula and could otherwise prevent these candidates appearing in PubChemLite queries at their true exact mass/formula. In the context of efficient screening of masses for environmental, metabolomics or exposomics studies, matches with differing isotopic states are unlikely to be found in large amounts in these studies. In the cases that isotopically labelled standards are used, or isotopically labelled experiments are performed, other data interrogation techniques are usually necessary/recommended to capture these peaks in advance of identification efforts. For differing charge states, since these are usually accounted for in the upstream workflow by adjusting the adduct state, the current behaviour ensures a consistent “base state” for adjustment of charge in other parts of the workflow. Secondly, mixtures are currently discarded from PubChemLite files, as this would require an additional degree of manipulation (splitting and re-merging of the entries), which was not accounted for in the current version as this affects < 10K entries - of which a significant proportion are salts. It would be possible to address both issues in future versions should subsequent use cases deem this necessary. Finally, related CIDs are only included if that CID contains any annotation in at least one of the selected annotation categories. Creating PubChemLite for MetFrag weekly following the January 14, 2020 format to test systems, with two versions used in this article to check additional annotation content (see results and [48]). During evaluations, it became clear that two additional categories would be useful, one being “Identification” (present but previously overlooked) and the second being “Associated Disorders and Diseases” (not present when PubChemLite was officially drafted). Based on the evaluations showing little difference between tier0 and tier1, one version equivalent to tier1 plus these two additiona categories has been built and released as “PubChemLite for exposomics” version 0.3.0 [39] and integrated into MetFragWeb [26] and patRoon [59, 63]. Subsequent updates will be built and auto-committed to Zenodo (after passing build checks) to allow automatic updates for MetFragWeb [26] and any workflows/users of the MetFrag command line (MetFragCL) version [58] and other workflows like patRoon [59]. Table 2 The breakdown of the major PubChemLite versions by InChIKey First Blocks (IKFB) and CIDs. 18 Nov 2019 14 Jan 2020 31 Oct 2020 tier0 tier1 tier0 tier1 exposomics PubChemLite (by IKFB) 315,842 361,556 316,810 363,911 371,663 Eliminated (by IKFB) 12,056 12,762 11,979 12,682 12,971 Total (by IKFB) 327,898 374,318 328,789 376,593 384,634 Parent CIDs (or CID, if no parent) 377,278 430,246 378,581 432,645 431,067 CIDs with desired annotation 402,746 458,621 405,285 462,356 462,838 For the November 18, 2019 versions, an “FPSum” was calculated for all entries by adding the FP bits to give a maximum of 7 (tier0) or 8 (tier1). Individual columns for each annotation category were also created, so that the annotation categories could be used via the scoring term function in MetFrag, in addition to the patent and literature information. The resulting datasets (with preview) are available on Zenodo [34]. For the January 14, 2020 and subsequent versions, “FPSum” was modified to “AnnoTotalCount”, so the column name better reflected the content, i.e. the availability of annotation categories for that entry. Additionally, individual columns were created for each annotation category, filled with values calculated by adding the category plus the number of subcategories present for that annotation, which ranged from 3 to 15 subcategories (Jan. 2020). The resulting datasets are on Zenodo [35] and were integrated into the dropdown menu of local databases for MetFragWeb [26]. PubChemLite was built approx. weekly following the January 14, 2020 format to test systems, with two versions used in this article to check additional annotation content (see results and [48]). Creating PubChemLite for MetFrag Subsequent updates will be built and auto-committed to Zenodo (after passing build checks) to allow automatic updates for MetFragWeb [26] and any workflows/users of the MetFrag command line (MetFragCL) version [58] and other workflows like patRoon [59]. Creating PubChemLite for MetFrag During evaluations, it became clear that two additional categories would be useful, one being “Identification” (present but previously overlooked) and the second being “Associated Disorders and Diseases” (not present when PubChemLite was officially drafted). Based on the evaluations showing little difference between tier0 and tier1, one version equivalent to tier1 plus these two additional categories has been built and released as “PubChemLite for exposomics” version 0.3.0 [39] and integrated into MetFragWeb [26] and patRoon [59, 63]. Subsequent updates will be built and auto-committed to Zenodo (after passing build checks) to allow automatic updates for MetFragWeb [26] and any workflows/users of the MetFrag command line (MetFragCL) version [58] and other workflows like patRoon [59]. For the November 18, 2019 versions, an “FPSum” was calculated for all entries by adding the FP bits to give a maximum of 7 (tier0) or 8 (tier1). Individual columns for each annotation category were also created, so that the annotation categories could be used via the scoring term function in MetFrag, in addition to the patent and literature information. The resulting datasets (with preview) are available on Zenodo [34]. For the January 14, 2020 and subsequent versions, “FPSum” was modified to “AnnoTotalCount”, so the column name better reflected the content, i.e. the availability of annotation categories for that entry. Additionally, individual columns were created for each annotation category, filled with values calculated by adding the category plus the number of subcategories present for that annotation, which ranged from 3 to 15 subcategories (Jan. 2020). The resulting datasets are on Zenodo [35] and were integrated into the dropdown menu of local databases for MetFragWeb [26]. PubChemLite was built approx. weekly following the January 14, 2020 format to test systems, with two versions used in this article to check additional annotation content (see results and [48]). During evaluations, it became clear that two additional categories would be useful, one being “Identification” (present but previously overlooked) and the second being “Associated Disorders and Diseases” (not present when PubChemLite was officially drafted). Based on the evaluations showing little difference between tier0 and tier1, one version equivalent to tier1 plus these two additiona categories has been built and released as “PubChemLite for exposomics” version 0.3.0 [39] and integrated into MetFragWeb [26] and patRoon [59, 63]. Creating PubChemLite for MetFrag Initial evaluations in this paper were done on the first archived versions, generated November 18th, 2019 [34], with 640 category fingerprints generated on October 2nd, 2019. There were approximately 33 M entries with TOC annotations at this stage (e.g. 33,766,782 on October 29th, 2019). A second archived version, with additional scoring, was created January 14th, 2020 [35] for further evaluation. By this time the fingerprint consisted of 652 categories (January 9th, 2020) and there were 35 M entries with TOC annotations (35,800,159 on 21 January 2020). The third major version, PubChemLite for exposomics (31 October, 2020) was based on a fingerprint of 524 categories (29 October 2020) and there were 49 M TOC annotations (49,493,641 on 2 November 2020). A breakdown of these files is given in Table 2. These datasets are archived as versions 0.1.0, 0.2.0 and 0.3.0 on Zenodo [34, 35, 39]. Page 10/25 Table 2 The breakdown of the major PubChemLite versions by InChIKey First Blocks (IKFB) and CIDs. 18 Nov 2019 14 Jan 2020 31 Oct 2020 tier0 tier1 tier0 tier1 exposomics PubChemLite (by IKFB) 315,842 361,556 316,810 363,911 371,663 Eliminated (by IKFB) 12,056 12,762 11,979 12,682 12,971 Total (by IKFB) 327,898 374,318 328,789 376,593 384,634 Parent CIDs (or CID, if no parent) 377,278 430,246 378,581 432,645 431,067 CIDs with desired annotation 402,746 458,621 405,285 462,356 462,838 For the November 18, 2019 versions, an “FPSum” was calculated for all entries by adding the FP bits to give a maximum of 7 (tier0) or 8 (tier1). Individual columns for each annotation category were also created, so that the annotation categories could be used via the scoring term function in MetFrag, in addition to the patent and literature nformation. The resulting datasets (with preview) are available on Zenodo [34]. For the January 14, 2020 and subsequent versions, “FPSum” was modified to “AnnoTotalCount”, so the column name better reflected the content, .e. the availability of annotation categories for that entry. Additionally, individual columns were created for each annotation category, filled with values calculated by adding the category plus the number of subcategories present for that annotation, which ranged from 3 to 15 subcategories (Jan. 2020). The resulting datasets are on Zenodo [35 and were integrated into the dropdown menu of local databases for MetFragWeb [26]. PubChemLite was built approx. Assessing PubChemLite The performance of PubChemLite was assessed using various datasets that were already used to evaluate MetFrag performance; CASMI 2016 [16] and MetFrag Relaunched [25] (hereafter MetFragRL). The CASMI2016 dataset consisted of 208 compound-MS/MS spectra pairs. The MetFragRL evaluation sets consisted of four groups of spectra measured under different conditions (datasets EA, EQEx, EQExPlus and UF, with n = 473, 289, 310 and 226, where n refers to the number of compound-MS/MS spectrum pairs). The calculations performed on the individual datasets are presented in Additional File 3, Table S1 and Figure S1, alongside the previously published results. Since some compounds had mass spectra available in both modes, and there was some overlap between the different datasets, this corresponded to a total of 1298 (MetFragRL) and 1506 (MetFragRL + CASMI) compound-MS/MS pairs overall. Calculations performed on this set (comparing PubChemLite tiers and CompTox) are presented in Additional File 3, Table S2 and Figure S2. For the purpose of clarity in the main manuscript, this set of 1506 was de-duplicated down to a set of 977 unique compounds by InChIKey First Block after accounting for multiple tautomeric forms, to eliminate any confusion due to the presence of duplicate spectra/modes. The MS/MS spectrum record number (the Page 11/25 Page 11/25 first-matching entry in the case of multiple spectra) was used to automatically extract and save the corresponding MS/MS peaks into the file using an R script, using the MS/MS spectra provided as SI for the respective studies, downloaded from the journal pages [16, 25]. As all compounds were present in PubChem, additional compound information was filled in using PubChem web services via R functions. The final benchmarking file (hereafter “PCLite Benchmark” set) is available as Additional File 2 and on the ECI GitLab pages, along with all associated code [61]. first-matching entry in the case of multiple spectra) was used to automatically extract and save the corresponding MS/MS peaks into the file using an R script, using the MS/MS spectra provided as SI for the respective studies, downloaded from the journal pages [16, 25]. As all compounds were present in PubChem, additional compound information was filled in using PubChem web services via R functions. The final benchmarking file (hereafter “PCLite Benchmark” set) is available as Additional File 2 and on the ECI GitLab pages, along with all associated code [61]. Assessing PubChemLite The PCLite Benchmark set was used to evaluate various versions of PubChemLite (dates: 18/11/2019 [34], 14/01/2020 [35], 22/05/2020 [48], 12/06/2020 [48] and 31/10/2020 [39]) as well as the CompTox Chemicals Dashboard version from 7/03/2019 archived as MetFrag Local CSV (database) files [38, 64]. Files are not yet available from the most recent CompTox release (but have been requested). The “Select Metadata” version of CompTox was used, which contained 857,615 entries, corresponding to 773,561 DTXCID InChIKeys and 773,232 InChIKey First Blocks associated with DTXCIDs (the information used in MetFrag). All CompTox files from the given release contain the same number of entries, just with varying metadata content. All queries were run with exact mass plus 5 ppm error, additional scoring terms and other parameters as detailed in Additional File 3, Table S4 and in the supporter scripts available on the ECI GitLab pages [65]. List Of Abbreviations List Of Abbreviations Page 12/25 API Application Programming Interface CASMI Critical Assessment of Small Molecule Identification CCS Collision Cross Section CID PubChem Compound Identifier CompTox US EPA CompTox Chemicals Dashboard DAG Directed Acyclic Graph DTXCID DSSTox Compound Identifier (from CompTox) DTXSID DSSTox Substance Identifier (from CompTox) ECI Environmental Cheminformatics group (at the University of Luxembourg) FPSum Addition of fingerprint bits to form a scoring term used in PubChemLite HMDB Human Metabolome Database IKFB InChIKey First Block KEGG Kyoto Encyclopedia of Genes and Genomes MetFragRL MetFrag Relaunched MoNA MassBank of North America MS/MS Tandem Mass Spectrum, MS2 NCBI National Center for Biotechnology Information NORMAN-SLE NORMAN Suspect List Exchange (NORMAN-SLE) PCL, PCLite PubChemLite RAM Random Access Memory SLE Suspect List Exchange (see NORMAN-SLE) TOC PubChem Compound Table of Contents (PubChem Compound TOC) TOC FP Table of Contents fingerprint (TOC FP) TOC Tree PubChem Compound Table of Contents (TOC) Tree ations Funding The work of EEB, PT, and JZ was supported by the Intramural Research Program of the National Library of Medicine, National Institutes of Health. ELS and TK acknowledge funding support from the Luxembourg National Research Fund (FNR) for project A18/BM/12341006. SN acknowledges BMBF funding under grant number 031L0107. Authors' contributions EEB & ELS conceptualized PubChemLite and annotation gap analysis; EEB coded PubChemLite files, ELS the evaluation, SN integrated into the MetFrag infrastructure. EEB, ELS discussed and developed the manuscript and concepts, SN contributed. PT developed the bit files; JZ, EEB, ELS and PT integrated the NORMAN-SLE files, transformation and annotation content into PubChem; TK implemented regular builds and associated infrastructure at LCSB. All authors have contributed to and approved the final manuscript. Availability of data and materials All the files needed to generate PubChemLite are available and updated at least weekly on the PubChem FTP website (https://ftp.ncbi.nlm.nih.gov/pubchem/) [60], all code to create PubChemLite with selected bit lists is available from the Environmental Cheminformatics group GitLab repository (https://git- r3lab.uni.lu/eci/pubchem/-/tree/master/pubchemlite) [61]. Fixed versions of PubChemLite mentioned in this manuscript are all archived on Zenodo [34, 35, 39, 48]. PubChemLite will be created and deposited to Zenodo at regular intervals, to allow integration with MetFrag [26], and offer download files for external users. The annotation Page 13/25 Page 13/25 content of the NORMAN-SLE (https://www.norman-network.com/nds/SLE/) [29] is being progressively added to PubChem [44], with all data available on PubChem [66] and Zenodo (https://zenodo.org/communities/norman-sle) [43]. The addition of new substances deposited to the NORMAN-SLE to PubChem is automated through mapping files and updated monthly (or more regularly if needed). content of the NORMAN-SLE (https://www.norman-network.com/nds/SLE/) [29] is being progressively added to PubChem [44], with all data available on PubChem [66] and Zenodo (https://zenodo.org/communities/norman-sle) [43]. The addition of new substances deposited to the NORMAN-SLE to PubChem is automated through mapping files and updated monthly (or more regularly if needed). Acknowledgements ELS acknowledges discussions with Rick Helmus (University of Amsterdam), Herbert Oberacher (Medical University of Innsbruck), Juliane Hollender (Eawag) and the Environmental Cheminformatics team (LCSB-ECI, University of Luxembourg). ELS & SN are grateful for the hard work of Christoph Ruttkies and Sebastian Wolf (both formerly IPB Halle) on MetFrag over the years that has enabled this work. 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EPA’s National Center For Computational Toxicology (2018) CompTox Chemicals Dashboard Metadata Files Integration with MetFrag. DOI: 10.23645/epacomptox.7525199.V1 64. EPA’s National Center For Computational Toxicology (2018) CompTox Chemicals Dashboard Metadata Files fo Integration with MetFrag. DOI: 10.23645/epacomptox.7525199.V1 65. Schymanski, Emma (2020) Environmental Cheminformatics GitLab Pages: PubChemLite R Script Folder. https://git-r3lab.uni.lu/eci/pubchem/-/tree/master/pubchemlite/R/. Accessed 27 Oct 2020 65. Schymanski, Emma (2020) Environmental Cheminformatics GitLab Pages: PubChemLite R Script Folder. 65. Schymanski, Emma (2020) Environmental Cheminformatics GitLab Pages: PubChemLite R Script Folder. https://git-r3lab.uni.lu/eci/pubchem/-/tree/master/pubchemlite/R/. Accessed 27 Oct 2020 66. NORMAN Network, NCBI/NLM/NIH (2020) NORMAN SLE Data Source in PubChem. https://pubchem.ncbi.nlm.nih.gov/source/23819. Accessed 7 May 2020 66. NORMAN Network, NCBI/NLM/NIH (2020) NORMAN SLE Data Source in PubChem. https://pubchem.ncbi.nlm.nih.gov/source/23819. References Accessed 7 May 2020 Figures Figure 1 PubChem Compound Table of Contents (TOC) Tree (2 Nov. 2020) from the PubChem Classification Browser [33]. The contents (and categories) are updated regularly. Left: the top 22 categories (of the current total 524) are shown (default view). Yellow shading indicates the seven categories used in PubChemLite tier0 (“environmental” selection the purple shading indicates the additional category used for PubChemLite tier1 (“exposomics”); red shading indicates the two categories that were added into the final PubChemLite exposomics selection. Right: Expansion of the “Agrochemical Information” and “Use and Manufacturing” sections as examples of sub-categories. Figure 1 PubChem Compound Table of Contents (TOC) Tree (2 Nov. 2020) from the PubChem Classification Browser [33]. The contents (and categories) are updated regularly. Left: the top 22 categories (of the current total 524) are shown (default view). Yellow shading indicates the seven categories used in PubChemLite tier0 (“environmental” selection), the purple shading indicates the additional category used for PubChemLite tier1 (“exposomics”); red shading indicates the two categories that were added into the final PubChemLite exposomics selection. Right: Expansion of the “Agrochemical Information” and “Use and Manufacturing” sections as examples of sub-categories. Page 18/25 Figure 1 PubChem Compound Table of Contents (TOC) Tree (2 Nov. 2020) from the PubChem Classification Browser [33]. The contents (and categories) are updated regularly. Left: the top 22 categories (of the current total 524) are show (default view). Yellow shading indicates the seven categories used in PubChemLite tier0 (“environmental” selection the purple shading indicates the additional category used for PubChemLite tier1 (“exposomics”); red shading indicates the two categories that were added into the final PubChemLite exposomics selection. Right: Expansion o the “Agrochemical Information” and “Use and Manufacturing” sections as examples of sub-categories. Figure 1 PubChem Compound Table of Contents (TOC) Tree (2 Nov. 2020) from the PubChem Classification Browser [33]. The contents (and categories) are updated regularly. Left: the top 22 categories (of the current total 524) are shown (default view). Yellow shading indicates the seven categories used in PubChemLite tier0 (“environmental” selection), the purple shading indicates the additional category used for PubChemLite tier1 (“exposomics”); red shading indicates the two categories that were added into the final PubChemLite exposomics selection. Right: Expansion of the “Agrochemical Information” and “Use and Manufacturing” sections as examples of sub-categories. Page 19/25 Page 19/25 Page 19/25 Figure 2 Sunburst plot of PubChemLite (14 January 2020 tier1 version [35]) to visualise the content. Figures Note many CIDs are in multiple sub-categories, and total counts include this duplication (i.e. the 5695 AgroChemInfo count corresponds with fewer unique CIDs, see below). An interactive version embedded in an RMarkdown file is available as Additional File 1, the interactive plot plus code and example file is also available on the ECI GitLab pages [36, 37]. Figure 2 Sunburst plot of PubChemLite (14 January 2020 tier1 version [35]) to visualise the content. Note many CIDs are in multiple sub-categories, and total counts include this duplication (i.e. the 5695 AgroChemInfo count corresponds with fewer unique CIDs, see below). An interactive version embedded in an RMarkdown file is available as Additional File 1, the interactive plot plus code and example file is also available on the ECI GitLab pages [36, 37]. Sunburst plot of PubChemLite (14 January 2020 tier1 version [35]) to visualise the content. Note many CIDs are in multiple sub-categories, and total counts include this duplication (i.e. the 5695 AgroChemInfo count corresponds with fewer unique CIDs, see below). An interactive version embedded in an RMarkdown file is available as Additional File 1, the interactive plot plus code and example file is also available on the ECI GitLab pages [36, 37]. Page 20/25 Figure 2 Sunburst plot of PubChemLite (14 January 2020 tier1 version [35]) to visualise the content. Note many CIDs are in multiple sub-categories, and total counts include this duplication (i.e. the 5695 AgroChemInfo count corresponds with fewer unique CIDs, see below). An interactive version embedded in an RMarkdown file is available as Additional File 1, the interactive plot plus code and example file is also available on the ECI GitLab pages [36, 37]. Figure 2 Figure 2 Sunburst plot of PubChemLite (14 January 2020 tier1 version [35]) to visualise the content. Note many CIDs are in multiple sub-categories, and total counts include this duplication (i.e. the 5695 AgroChemInfo count corresponds with fewer unique CIDs, see below). An interactive version embedded in an RMarkdown file is available as Additional File 1, the interactive plot plus code and example file is also available on the ECI GitLab pages [36, 37]. Sunburst plot of PubChemLite (14 January 2020 tier1 version [35]) to visualise the content. Note many CIDs are in multiple sub-categories, and total counts include this duplication (i.e. the 5695 AgroChemInfo count corresponds with fewer unique CIDs, see below). An interactive version embedded in an RMarkdown file is available as Additional File 1, the interactive plot plus code and example file is also available on the ECI GitLab pages [36, 37]. Page 21/25 Figure 3 Figure 3 Figure 3 Page 21/25 The ranking performance of various versions of PubChemLite versus CompTox using the merged benchmarking set (n=977) with comparable metadata terms. Green: without MS/MS information. Blue: with MS/MS information (includes in silico fragmentation and MoNA library scoring terms). The increase in top ranks and decrease in missing entries with newer versions shows the influence of additional annotation content in PubChem (see Section “Filling Annotation Gaps”). The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Figure 3 The ranking performance of various versions of PubChemLite versus CompTox using the merged benchmarking set (n=977) with comparable metadata terms. Green: without MS/MS information. Blue: with MS/MS information (includes in silico fragmentation and MoNA library scoring terms). The increase in top ranks and decrease in missing entries with newer versions shows the influence of additional annotation content in PubChem (see Section “Filling Annotation Gaps”). The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Page 23/25 Figure 5 Page 23/25 Figure 5 Figure 3 The ranking performance of various versions of PubChemLite versus CompTox using the merged benchmarking set (n=977) with comparable metadata terms. Green: without MS/MS information. Blue: with MS/MS information (includes in silico fragmentation and MoNA library scoring terms). The increase in top ranks and decrease in missing entries with newer versions shows the influence of additional annotation content in PubChem (see Section “Filling Annotation Gaps”). The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. The ranking performance of various versions of PubChemLite versus CompTox using the merged benchmarking set (n=977) with comparable metadata terms. Green: without MS/MS information. Blue: with MS/MS information (includes in silico fragmentation and MoNA library scoring terms). The increase in top ranks and decrease in missing entries with newer versions shows the influence of additional annotation content in PubChem (see Section “Filling Annotation Gaps”). The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Page 22/25 Figure 4 Screenshot of the NORMAN Suspect List Exchange Classification in PubChem (13 August 2020), including (partial) expansions of the S60, S66 and S68 lists, with the corresponding sections added to individual records indicated in green type. Figure 4 Screenshot of the NORMAN Suspect List Exchange Classification in PubChem (13 August 2020), including (partial) expansions of the S60, S66 and S68 lists, with the corresponding sections added to individual records indicated in green type. Figure 4 Screenshot of the NORMAN Suspect List Exchange Classification in PubChem (13 August 2020), including (partial) expansions of the S60, S66 and S68 lists, with the corresponding sections added to individual records indicated in green type. Screenshot of the NORMAN Suspect List Exchange Classification in PubChem (13 August 2020), including (partial) expansions of the S60, S66 and S68 lists, with the corresponding sections added to individual records indicated in green type. Figure 5 Transformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [45]. ransformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [45]. Figure 5 Page 23/25 Transformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [45]. Figure 5 Transformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [45]. Figure 6 Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotatio MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live que 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third of the AgroChemScore (number of subcategories of agrochemical information available). The Ag (yet) available for the full database. Note: missing agrochemical entries are due to the presence o Transformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [45]. Transformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [4 Figure 5 Transformations section in PubChem for Folpet (CID 8607) from SWISSPEST19 [45]. Figure 6 Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotation score alone (no MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live query, 22 Jan 2020 with 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third: with the addition of the AgroChemScore (number of subcategories of agrochemical information available). The AgroChemScore is not (yet) available for the full database. Note: missing agrochemical entries are due to the presence of metals in some agrochemicals, which are excluded from MetFrag results (see Methods for rules applied to create PubChemLite). Bottom in blue shading: Ranking performance of PubChemLite (12 Jun. 2020) in MetFrag using topic-specific annotation score plus MS/MS information on the subsets of the benchmarking containing agrochemical annotation Figure 6 Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotation score alone (no MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live query, 22 Jan 2020 with 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third: with the addition of the AgroChemScore (number of subcategories of agrochemical information available). The AgroChemScore is not (yet) available for the full database. Note: missing agrochemical entries are due to the presence of metals in some agrochemicals, which are excluded from MetFrag results (see Methods for rules applied to create PubChemLite). Bottom in blue shading: Ranking performance of PubChemLite (12 Jun. 2020) in MetFrag using topic-specific annotation score plus MS/MS information on the subsets of the benchmarking containing agrochemical annotation information. The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotation score alone (no MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live query, 22 Jan 2020 with 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third: with the addition of the AgroChemScore (number of subcategories of agrochemical information available). The AgroChemScore is not (yet) available for the full database. Note: missing agrochemical entries are due to the presence of metals in some agrochemicals, which are excluded from MetFrag results (see Methods for rules applied to create PubChemLite). Bottom in blue shading: Ranking performance of PubChemLite (12 Jun. 2020) in MetFrag using topic-specific annotation score plus MS/MS information on the subsets of the benchmarking containing agrochemical annotation information. The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Figure 6 2020) in MetFrag using topic-specific annotation score plus MS/MS information on the subsets of the benchmarking containing agrochemical annotation information. The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. information. The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42] information. The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Figure template from [42]. Figure 6 Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotation score alone (no MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live query, 22 Jan 2020 with 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third: with the addition of the AgroChemScore (number of subcategories of agrochemical information available). The AgroChemScore is not (yet) available for the full database. Note: missing agrochemical entries are due to the presence of metals in some agrochemicals, which are excluded from MetFrag results (see Methods for rules applied to create PubChemLite). Bottom in blue shading: Ranking performance of PubChemLite (12 Jun. 2020) in MetFrag using topic-specific annotation score plus MS/MS information on the subsets of the benchmarking containing agrochemical annotation Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotation score alone (no MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live query, 22 Jan 2020 with 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third: with the addition of the AgroChemScore (number of subcategories of agrochemical information available). The AgroChemScore is not (yet) available for the full database. Note: missing agrochemical entries are due to the presence of metals in some agrochemicals, which are excluded from MetFrag results (see Methods for rules applied to create PubChemLite). Bottom in blue shading: Ranking performance of PubChemLite (12 Jun. 2020) in MetFrag using topic-specific annotation score plus MS/MS information on the subsets of the benchmarking containing agrochemical annotation Page 24/25 Page 24/25 information. The script and associated data files to reproduce this plot are available on the ECI GitLab pages [40, 41]. Figure template from [42]. Figure 6 Green shading: Ranking performance of PubChemLite (14 Jan. 2020) in MetFrag using annotation score alone (no MS/MS information) with the Agrochemicals set from 14 Jan 2020. Top: full PubChem (live query, 22 Jan 2020 with 102,404,298 compounds). Second: PubChemLite tier1 with literature and patent scores and third: with the addition of the AgroChemScore (number of subcategories of agrochemical information available). The AgroChemScore is not (yet) available for the full database. Note: missing agrochemical entries are due to the presence of metals in some agrochemicals, which are excluded from MetFrag results (see Methods for rules applied to create PubChemLite). Bottom in blue shading: Ranking performance of PubChemLite (12 Jun. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download PubChemLiteSIAdditionalFile3noRefs.pdf PubChemLiteSIAdditionalFile3noRefs.pdf PubChemLiteSIAdditionalFile3noRefs.pdf Page 25/25 Page 25/25 Page 25/25
https://openalex.org/W4211098969	http://pteo.uniza.sk/doi/10.26552/pte.C.2008.1.7.pdf	Slovak	null	Diverzifikácia portfólia	Pošta, telekomunikácie a elektronický obchod	2,008	cc-by	2,081	Pošta, Telekomunikácie a Elektronický obchod Pošta, Telekomunikácie a Elektronický obchod ISSN 1336-8281 + Ing. Lucia Tokarčíková, externá doktorandka na Katedre spojov, Žilinskej univerzity v Žiline, Slovenská republika Lucia Tokarčíková+ Diverzifikácia je jav, ktorý vzniká pri kombinácii aktív, ktorých výnosy majú korelačný koeficient nižší ako 1. V praxi to znamená, že takouto kombináciou investor dosiahne značné zníženie rizika v porovnaní s priemerným rizikom týchto aktív osobitne pri zachovaní priemerného očakávaného výnosu. Diverzifikácia portfólia je asi najzákladnejším problémom investora. Ideálna diverzifikácia by znamenala nákup všetkých rizikových aktív, ktoré trh ponúka, pretože by takto investor úplne odstránil špecifické riziko. Ostalo by len trhové riziko, ktoré by mohlo ovplyvniť výnos jeho portfólia. Keďže väčšinou z rôznych dôvodov nie je možné takúto náročnú investíciu realizovať bude sa musieť investor uspokojiť len s kombináciou niektorých aktív. Aby odstránil špecifické riziko, investor by mal kombinovať také aktíva, ktoré majú zápornú koreláciu odchýlok od očakávaného výnosu. V praxi sa však v súčasnosti sotva stretneme s dvoma aktívami, ktorých výnosy sú záporne korelované, preto sa investor bude musieť uspokojiť aj s kladnou koreláciou, avšak vždy menšou ako 1. V opačnom prípade by diverzifikácia nemala zmysel, pretože celkové riziko by už nebolo výsledkom kovariancie výnosov týchto aktív, ale len ich aritmetického priemeru váženého podielom jednotlivých aktív v portfóliu. Efektívne portfólio je také aktívum alebo kombinácia aktív, ktoré má medzi aktívami z rizikovej triedy najvyšší očakávaný výnos. Hľadanie efektívnych portfólií a ich konštrukcie je náplňou portfóliového manažmentu. Bez ohľadu na to aký sklon k riziku má investor, musí platiť nasledujúci princíp, ktorý Sharpe nazýva „vetou o efektívnej množine.“ V efektívnej množine investor si vyberie svoje optimálne portfólio z množiny portfólií, ktoré: 1. ponúkajú maximálny očakávaný výnos pri rôznych úrovniach rizika, 1. ponúkajú maximálny očakávaný výnos pri rôznych úrovniach rizika 2. ponúkajú minimálne riziko pri rôznych úrovniach očakávaného výn 2. ponúkajú minimálne riziko pri rôznych úrovniach očakávaného výnosu. To znamená, že racionálny investor sa môže rozhodnúť investovať len do takej komibnácie aktív, ktorá leží na efektívnej množine. Efektívna množina je podmnožinou prípustnej množiny. Efektívnu množinu kombinácie akcií ACDB znázorňuje úsek PB. Úsek PA nepatrí do efektívnej množiny, lebo pri rovnakom riziku sa dá dosiahnuť vyšší výnos. prípustnej množiny. Efektívnu množinu kombinácie akcií ACDB znázorňuje úsek PB. Úsek PA nepatrí do efektívnej množiny, lebo pri rovnakom riziku sa dá dosiahnuť vyšší výnos. 43 I/2008 43 Pošta, Telekomunikácie a Elektronický obchod ISSN 1336-8281 ISSN 1336-8281 ISSN 1336-8281 Obrázok č. 1 Prípustná a efektívna množina Obrázok č. 1 Prípustná a efektívna množina Z uvedeného vyplýva, že prostriedky musíme rozdeliť takým spôsobom, aby takáto kombinácia vytvorila portfólio, ktoré leží v úseku PB, napríklad portfólio O. Lucia Tokarčíková+ Pomer jednotlivých inštrumentov v portfóliu určíme pomocou úlohy kvadratického programovania. Kvadratické programovanie je úloha matematického programovania, ktorého ohraničenia sú lineárne a ktorého účelová funkcia je nelineárna suma výrazov x1 k1 + x2 k2+...+ xn kn so stupňom k1+k2+...+kn, maximálne 2. Bodu O zodpovedá výnos ro Smerodajnej odchýlke σo zodpovedá nekonečne veľa očakávaných výnosov vo vnútri prípustnej množiny, napríklad aj výnos rσ, ktorý je pre danú odchýlku najmenší. Hľadá sa však iba ten bod, ktorý je zároveň aj súčasťou efektívnej množiny. Pri danom očakávanom výnose je teda potrebné nájsť také pomery xi, ktoré minimalizujú smerodajnú odchýlku. Účelová funkcia takejto úlohy je matematicky vyjadrená nasledovne: ∑∑ = = N j ij j i x x 1 1 σ = N i p Min σ (1.) (1.) Výnos celého portfólia sa musí rovnať priemeru výnosov jednotlivých akcií vážených ich podielom v portfóliu: Výnos celého portfólia sa musí rovnať priemeru výnosov jednotlivých akcií vážených ich podielom v portfóliu: ∑ = = N i o i i r r x 1 (1.2) (1.2) Súčet podielov jednotlivých akcií sa musí rovnať 100%: Súčet podielov jednotlivých akcií sa musí rovnať 100%: ∑ = = N i i x 1 1 , (1.3) (1.3) a podiel akcií musí byť nezáporný, aby sa predišlo riešeniu s predajom nakrátko: a podiel akcií musí byť nezáporný, aby sa predišlo riešeniu s predajom nakrátko: 0 ≥ ix ; i=1,2,...,N. Predchádzajúce tri vzťahy sú lineárnymi podmienkami optimalizačnej úlohy. Takýto postup zaručí, že vypočítané portfólio, bude ležať na efektívnej množine. Na základe vstupných údajov je zrejmé, že pri väčšom počte cenných papierov v portfóliu je potrebné vyrátať veľké množstvo kovariancií. I/2008 44 44 Pošta, Telekomunikácie a Elektronický obchod ISSN 1336-8281 Portfólio musí v každom prípade ležať v efektívnej množine. Vieme ako vypočítať podiel jednotlivých aktív pomocou kvadratického programovania. Všetky portfólia, ktoré ležia v efektívnej množine sú najlepšie, aké si môže investor vybrať. Problém je, že ich je nekonečne veľa, preto si musí vybrať len jedno. Ako určiť, ktoré portfólio je optimálne? Aby sme vedeli odpovedať, musíme si zaviesť pojem averzie investora k riziku a jej grafické znázornenie indiferenčnou krivkou. Krivky indiferencie reprezentujú investorove preferencie rizika a výnosnosti. Mapa kriviek indiferencie je vlastná hypotetickému investorovi. Každá krivka indiferencie reprezentuje všetky kombinácie portfólií, ktoré by investor považoval za rovnako žiadúce. Všeobecne vieme, že ak sú dané dve portfólia s rovnakou smerodajnou odchýlkou, potom si vyberie portfólio s vyššou očakávanou výnosnosťou. Lucia Tokarčíková+ Ako sa však bude správať investor pri výbere portfólia s rovnakou výnosnosťou, ale s rôznou veľkosťou smerodajných odchýlok. Závisí to od toho, aký má investor odpor k riziku. Niektorý investori majú vysoký, iní zase len mierny odpor k riziku. Investori podľa toho budú mať aj rôzne mapy kriviek indiferencie. Obrázok č. 2 Krivky indiferencie investora Obrázok č. 2 Krivky indiferencie investora Portfólia „A“ a „B“ sú rovnako žiadúce. „B“ má väčšiu smerodajnú odchýlku, ale túto väčšiu smerodajnú odchýlku kompenzuje väčšia očakávaná výnosnosť. Portfólio „C“ leží na krivke indiferencie, ktorá je žiadúcejšia než portfólia „A“ a „D“. I3 je viac žiadúcejšie než I2. „C“ má skoro také isté riziko ako „D“, ale je vyššie, či je výnosnejšie. Krivky indiferencie sa nemôžu pretínať. Je ich nekonečne veľa, investor bude považovať za žiadúcejšie ľubovoľné portfólio, ktoré leží na krivke indiferencie, ktorá je umiestnená vyššie než iné krivky indiferencie (I4, I5). Krivky indiferencie sa nikdy nepretínajú. Obrázok č. 2 Krivky indiferencie investora Portfólia „A“ a „B“ sú rovnako žiadúce. „B“ má väčšiu smerodajnú odchýlku, ale túto väčšiu smerodajnú odchýlku kompenzuje väčšia očakávaná výnosnosť. Portfólio „C“ leží na krivke indiferencie, ktorá je žiadúcejšia než portfólia „A“ a „D“. I3 je viac žiadúcejšie než I2. „C“ má skoro také isté riziko ako „D“, ale je vyššie, či je výnosnejšie. Krivky indiferencie sa nemôžu pretínať. Je ich nekonečne veľa, investor bude považovať za žiadúcejšie ľubovoľné portfólio, ktoré leží na krivke indiferencie, ktorá je umiestnená vyššie než iné krivky indiferencie (I4, I5). Krivky indiferencie sa nikdy nepretínajú. Krivky indiferencie sa stanovujú pomocou teórie hazardných hier, pri ktorej sa využívajú dva predpoklady: ť: ak si investor môže vybrať medzi dvoma inak zhodnými portfóliami vyberie si také, ktoré má vyššiu očakávanú výnosnosť. Investori budú dávať prednosť vyššej úrovni koncového bohatstva 2. odpor k riziku: investori sa vyhýbajú riziku k hazardu, pretože potenciálne straty predstavujú nepríjemnosti, ktoré sú vyššie než je veľkosť potešenia z investovania Investori si vyberajú medzi dvoma zhodnými portfóliami predovšetkým také, ktoré majú vyššiu očakávanú výnosnosť, ak sa však nebudú môcť rozhodnúť podľa očakávaných 2. Lucia Tokarčíková+ odpor k riziku: investori sa vyhýbajú riziku k hazardu, pretože potenciálne straty predstavujú nepríjemnosti, ktoré sú vyššie než je veľkosť potešenia z investovania Investori si vyberajú medzi dvoma zhodnými portfóliami predovšetkým také, ktoré majú vyššiu očakávanú výnosnosť, ak sa však nebudú môcť rozhodnúť podľa očakávaných straty predstavujú nepríjemnosti, ktoré sú vyššie než je veľkosť potešenia z investovania Investori si vyberajú medzi dvoma zhodnými portfóliami predovšetkým také, ktoré majú vyššiu očakávanú výnosnosť, ak sa však nebudú môcť rozhodnúť podľa očakávaných Investori si vyberajú medzi dvoma zhodnými portfóliami predovšetkým také, ktoré majú vyššiu očakávanú výnosnosť, ak sa však nebudú môcť rozhodnúť podľa očakávaných I/2008 45 ISSN 1336-8281 Pošta, Telekomunikácie a Elektronický obchod výnosností pristupuje ďalšie kritérium a to veľkosť smerodajnej odchýlky, kde sa potom rozhodujú podľa veľkosti týchto odchýliek. Predpokladá sa, že investori majú odpor k riziku, teda mali by si vyberať portfólio s menšou smerodajnou odchýlkou. výnosností pristupuje ďalšie kritérium a to veľkosť smerodajnej odchýlky, kde sa potom rozhodujú podľa veľkosti týchto odchýliek. Predpokladá sa, že investori majú odpor k riziku, teda mali by si vyberať portfólio s menšou smerodajnou odchýlkou. Investor môže mať jeden z troch postojov k riziku: - odpor k riziku - vyhľadávanie rizika - rizikovo neutrálny investor Krivky indiferencie u investorov s odporom k riziku Krivky indiferencie u investorov s odporom k riziku 1. Investor, ktorý má odpor k riziku – krivky majú sklon konvexný, sklon rastie zľava doprava. Tento odpor môže byť: a) vysoký b) neutrálny c) nízky Obrázok č. 3 Investor s vysokým odporom k riziku r, ktorý má odpor k riziku – krivky majú sklon konvexný, s . Tento odpor môže byť: a) vysoký b) neutrálny c) nízky Obrázok č. 3 Investor s vysokým odporom k riziku 1. Investor, ktorý má odpor k riziku – krivky majú sklon konvexný, sklon rastie zľava doprava. Tento odpor môže byť: a) vysoký b) neutrálny c) nízky 1. Investor, ktorý má odpor k riziku – krivky majú sklon konvexný, sklon rastie zľava doprava. Tento odpor môže byť: a) vysoký b) neutrálny c) nízky odpor môže byť: a) vysoký b) neutrálny c) nízky Obrázok č. 3 Investor s vysokým odporom k riziku Investor s odporom k riziku pre investovanie vyberie na investovanie portfólio, ktoré leží na „najvyššie vľavo“ položenej krivke indiferencie, t.j. pri voľbe medzi dvoma investíciami rovnakým predpokladaným výnosom bude vyberať menšie riziko. Obrázok č. 4 Investor s neutrálnym vzťahom k riziku Obrázok č. Lucia Tokarčíková+ 4 Investor s neutrálnym vzťahom k riziku Investor s neutrálnym vzťahom k riziku je k riziku indiferentný a pri svojich investičných rozhodnutiach neberie do úvahy. Obrázok č. 4 Investor s neutrálnym vzťahom k riziku Investor s neutrálnym vzťahom k riziku je k riziku indiferentný a pri svojich investičných rozhodnutiach neberie do úvahy. I/2008 46 ISSN 1336-8281 Pošta, Telekomunikácie a Elektronický obchod Obrázok č. 5 Investor s nízkym odporom k riziku Obrázok č. 5 Investor s nízkym odporom k riziku Nakoniec je investor, ktorý riziko vyhľadáva a ak má si vybrať medzi dvoma investíciami s rovnakým výnosom, potom investuje tam, kde je vyššie riziko. Sklon kriviek indiferencie závisí od veľkosti koeficienta absolútneho odporu k riziku alebo na jeho prevrátenej hodnote, t.j. koeficientu tolerancie, ktorý je vždy kladný. N N N N N σ2 = [rp - E (rp )]2 = E ∑ [ri - E (ri )]2 . wi = ∑ ∑ wi wj σij = ∑ ∑ wi wj σi σj ρij (1.4) i=1 i=1 j=1 i=1 j=1 (1.4) kde σij – je kovariancia medzi výnosom z i-tého a j-tého cenného papiera kde σij – je kovariancia medzi výnosom z i-tého a j-tého cenného papiera kde σij – je kovariancia medzi výnosom z i-tého a j-tého cenného papiera ρij – korelácia medzi výnosmi i-tého a j-tého cenného papiera j ρij – korelácia medzi výnosmi i-tého a j-tého cenného papiera j ρij – korelácia medzi výnosmi i-tého a j-tého cenného papiera Táto kovariancia vo vzťahu k smerodajne odchýlke i-tého a odchýlke j-tého cenného papieru nám vyjadruje koreláciu medzi výnosmi uvedených cenných papierov. Môžeme to jednoducho vyjadriť ako vzťah: COVi,j ρij = –––––––– (1.5) σi σj (1.5) kde COVi,j - kovariancia výnosov medzi akciami i,j σi σj – štandardná odchýlka výnosu kde COVi,j - kovariancia výnosov medzi akciami i,j σi σj – štandardná odchýlka výnosu kde COVi,j - kovariancia výnosov medzi akciami i,j σi σj – štandardná odchýlka výnosu kde COVi,j - kovariancia výnosov medzi akciami i,j σi σj – štandardná odchýlka výnosu kde COVi,j - kovariancia výnosov medzi akciami i,j σi σj – štandardná odchýlka výnosu Výhody diverzifikácie spočívajú v tom, že vhodnou kombináciou aktív, ktorých korelácia nedosahuje až formou úplne kladnej korelácie, sa dosiahne účinnejší kompenzačný efekt rizika a výnosu. V takýchto prípadoch je štandardná odchýlka výnosu portfólia menšia ako vážený priemer štandardných odchýlok aktív v portfóliu. Diverzifkácia znižuje riziko aj pri menšom počte cenných papierov – najskôr znižuje rýchlo, postupne so zvyšujúcim sa množstvom cenných papierov, účinnosť klesá. Korelácia výnosov má zásadný význam pre určenie štandardnej odchýlky portfólia. S narastajúcim počtom cenných papierov v portfóliu sa zoslabuje vplyv variácií jednotlivých aktív. I/2008 47 Pošta, Telekomunikácie a Elektronický obchod ISSN 1336-8281 Literatúra [1] CHOVANCOVÁ B., JANKOVSKÁ A., KOTLEBOVÁ J., ŠTURC B.: Finančný trh, Bratislava, EUROUNION, 2002, ISBN 80-88984-31-9 [2] SHARPE, W. F. – ALEXANDER, G. J.: Investice, Praha, Victoria Publishing, l994, ISBN 80-85605-47-3 Pošta, Telekomunikácie a Elektronický obchod ISSN 1336-8281 ISSN 1336-8281 Literatúra [1] CHOVANCOVÁ B., JANKOVSKÁ A., KOTLEBOVÁ J., ŠTURC B.: Finančný trh, Bratislava, EUROUNION, 2002, ISBN 80-88984-31-9 [2] SHARPE, W. F. – ALEXANDER, G. J.: Investice, Praha, Victoria Publishing, l994, ISBN 80-85605-47-3 I/2008 48 48
W4309602878.txt	https://conjecturas.org/index.php/edicoes/article/download/1942/1429	pt		Avaliação da adição de serina no diluente para aprimoramento da conservação de sêmen canino em baixas temperaturas	Conjeturas	2,022	cc-by	4,155	DOI: 10.53660/CONJ-1942-2R55A Avaliação da adição de serina no diluente para aprimoramento da conservação de sêmen canino em baixas temperaturas Evaluation of the addition of serine in the diluent for canine semen conservation improvement in low temperatures Autor Maria Eduarda Bicca Dode1, Carine Dahl Corcini¹, Edenara Anastácio da Silva¹,Stela Meneghello Gheller¹, Antônio Sérgio Varela Jr2. RESUMO A conservação de sêmen canino através da refrigeração promove alterações celulares capazes de interferir na viabilidade dos espermatozóides. O objetivo deste estudo foi avaliar o efeito da adição de serina no meio diluidor sobre a cinética espermática do sêmen canino resfriado. Foram realizadas 12 coletas, utilizando 4 cães da raça Australian Cattle Dog sendo ejaculados com motilidade superior a 60% e vigor ≥3. Foram testados diluentes Tris gema 20% (TG) com adição nove concentrações diferentes de serina (S) de 2,5mM a 200mM de S. Um grupo sem aminoácido foi utilizado como controle (TG0). Foram analisados no “Computerized-assisted sperm motility analysis” dos parâmetros de cinética nas horas zero, 24, 48 e 72 de resfriamento a 4° C. A adição de serina em concentrações de até 5 mM são promissoras para uso em diluentes de sêmen canino visando manter os parâmetros de motilidade espermática, sendo o potencial protetor da adição de serina em diluentes sêmen canino é dose e tempo dependente. Palavras-chave: Aminoácido 1; Cão 2; Resfriamento 3; ABSTRACT Canine semen conservation through refrigeration promotes cellular changes capable of interfering with sperm viability. The aim of this study was to evaluate the effect of adding serine to the extender medium on the sperm kinetics of cooled canine semen. Twelve collections were performed, using 4 Australian Cattle Dogs, which were ejaculated with motility greater than 60% and vigor ≥3. 20% Tris yolk (TG) extenders were tested with the addition of nine different concentrations of serine (S) from 2.5mM to 200mM of S. A group without amino acid was used as a control (TG0). The kinetic parameters were analyzed in the "Computerized-assisted sperm motility analysis" at 0, 24, 48 and 72 hours of cooling at 4°C. The addition of serine in concentrations up to 5 mM are promising for use in canine semen extenders. aiming to maintain the parameters of sperm motility, being the protective potential of the addition of serine in canine semen extenders is dose and time dependent. Keywords: Aminoacid 1; Dog 2; Cooling 3; Instuição de afiliação Universidade Federal de Pelotas. E-mail: dudadode@hotmail.com 2 Instuição de afiliação Universidade Federal de Rio Grande. 1 Conjecturas, ISSN: 1657-5830, Vol. 22, Nº 15 INTRODUÇÃO A cinofilia vem avançando e exigindo da medicina veterinária suporte científico e tecnológico capaz de suprir a crescente demanda de criadores e proprietários pelas biotecnicas reprodutivas. O aprimoramento de metodologias aplicáveis à caninos é o reflexo da relevância econômica e social dos cães na sociedade contemporânea pois além do valor zootécnico e de aptidões e habilidades específicas os cães podem ser utilizados como modelo experimental para outras espécies inclusive algumas ameaçadas de extinção (Pena, 2006), como Lobo Vermelho (Canis rufus), o Lobo da Etiópia (Canis simensis), o Cão de Caça Africano (Licaon pictus) bem como para espécies brasileiras, como o Lobo Guará (Chrysocyonbrachyurus), a Raposinha do Campo (Pseudalopex sp.)e o Cachorro Vinagre (Speothos venaticus)(Silva et al, 2000). A técnica de resfriamento de sêmen promove a preservação temporária de gametas em baixas temperaturas, sem que se atinja o estado de quiescência. Contudo, o processo predispõe alterações intracelulares nocivas que interferem na viabilidade dos espermatozoides (Iguer-Ouada, 2001). A resistencia térmica dos espermatozoides distingue-se entre as espécies, sendo os equinos, felinos, canideos e humanos pouco sensíveis (Watson & Plummer, 1985; Bwanga, 1991), visto que o componente fosfolipidico da membrana celular dessas espécies proporciona uma maior estabilidade celular (Bouchard Et Al., 1990). Manipulações no semen incluem resfriamento, congelação e descongelação dos espermatozoides e provocam mudanças na estrutura da bicamada lipidica da membrana, fazendo com que algumas proteínas livres possam se ligar a outras substancias (Rodrigues-Martinez et al., 1993).A redução da temperatura amplia o período de armazenamento do sêmen contribuindo para ampliar o período de viabilidade mas, ainda assim, há risco de alterações na estrutura e fisiologia celular devido ao choque térmico, danos mecânicos, estresses químicos e osmóticos do processo de conservação ( Khalili, 2010). Acredita-se que a cada 10ºC. de redução de temperatura, o metabolismo seja reduzido em 50%, sendo que a temperatura ideal para armazenamento a curto prazo é variável devido a composição dos lipídeos das membranas. Para obter sucesso no resfriamento é necessário que o meio de diluição previna danos, permita a manutenção do pH, potencial iônico e osmolaridade, e contenha fonte energética (England, 1993). A manutenção da fertilidade do sêmen que sofreu choque 1115 térmico ainda é fator limitante em ovinos, caprinos e caninos e se deve aos danos causados pelo choque térmico, exposição a temperaturas baixas, estresse osmótico e oxidativo de forma isolada ou combinada (BEHERA et al., 2015). Mesmo que se almeje apenas a conservação a curto prazo do semen sob refrigeração, meios diluidores adequados devem ser utilizados. (Santos et al., 2002). Aminoácidos com cadeias laterais polares desprovidos de carga apresentam carga liquida igual a zero em pH neutro e assim como a treonina e a tirosina, a serina (Ser ou S) possui um grupamento hidroxila capaz de estabelecer pontes de hidrogênio. A a cadeia lateral da serina participa como componente importante do sitio ativo em diversas enzimas (Champe et al., 2005; Nelson e COX, 2008). A fosfatidil-serina é um fosfolipídeo de membrana que tem sua porção hidrofílica constituída por um grupamento fosfato ligado à serina. A fosfatidil-serina é necessária para a síntese das membranas celulares em mamíferos. A síntese de fosfatidil-serina ocorre através da troca de bases reversível, onde a etanolamina é substituída por serina livre (Champe et al, 2005).Nem todos os autores relataram o efeito positivo da adição de AA no meio de resfriamento de semen, altas concentrações tornam-se prejudiciais pois induzem uma elevação da pressão osmótica causando danos celulares (Uysal, O., Buck, M.N., 2007, Pena, 1998) Visto às diferenças relativas a espécie e aos indivíduos, para um maior aproveitamento do sêmen é necessário que as técnicas de conservação sejam estabelecidas e ajustadas ampliando as possibilidade de aplicação de biotécnicas reprodutivas de forma eficiente.O objetivo deste estudo foi avaliar o efeito imediato da adição de meio diluidor com prolina sobre a motilidade espermática e seu efeito na longevidade e motilidade do sêmen canino diluído e resfriado através da sistema “Computerized-assisted sperm motility analysis” (CASA (SpermVision®, Minitube, Tiefenbach, Alemanha). MATERIAL E MÉTODOS Neste estudo os ejaculados utilizados foram obtidos de 12 coletas, realizadas em 4 cães da raça Australian Cattle Dog (1 - 3 anos), de propriedade do Canil Sentinela Farrapo, clinicamente saudáveis, com fertilidade comprovada após monta natural e rotina 1116 de coleta seminal. Todos os animais foram mantidos com as mesmas condições de alimentação e manejo. No experimento apenas ejaculados que apresentassem motilidade superior a70% e vigor ≥3 foram utilizados. Para elaboração dos meios e diluentes todos os reagentes utilizados neste experimento foram provenientes da Sigma-Aldrich® (St. Louis, MO, USA) e o diluente composto de Tris gema 20% (TG0) (Varela Junior et al., 2009) sem aminoácidos foi utilizado como controle. Foram testados diluentes com nove concentrações diferentes de Serina (S): TG+ 200mM S (TG200S), TG+ 100mMS (TG100S), TG+ 50mMS (TG50S), TG+ 40mM S (TG40S), TG+ 30mM S (TG30S), TG+ 20mM S (TG20S), TG+ 10mM S (TG10S), TG+ 5mM S (TG5S) e TG+ 2,5mMS (TG2,5S). As coletas de semen foram realizadas por manipulação digital e a freqüência de coleta dos cães foi de duas vezes por semana. Imediatamente após a coletafoi avaliado o volumee a concentração do ejaculado. Volume foi mensurado utilizando micropipetadora. Concentração foi determinada através da câmara de Neubauer. Os ejaculados foram então fracionados entre os 10 tratamentos e entre os quatro momentos de avaliações que seriam realizados (0h, 24h, 48h e 72h), e diluídos nos tratamentos a temperatura de 34° C na concentração finalde 10x107 de espermatozoides/mL-1. A curva de resfriamento foi realizada em caixa condicionadora (Koolmate, Minitube, Ge), com taxa de resfriamento de 0,3-0,5°C.min-1 até atingir a temperatura de 5°C, permanecendo as amostras armazenadas durante 72h. A motilidade espermática foi avaliada através sistema CASA em um microscópio óptico (Axio Scope A1®, Zeiss, Jena, Alemanha) a 200 X.). O sêmen previamente diluído nos tratamentos e refrigerado foi aquecido a 37°C durante 10 minutos antes da analise de motilidade que utilizou uma alíquota de 10 μL de sêmen, avaliada entre lâmina e lamínula. As variáveis analisadas pelo sistema foram: Motilidade total (MT) e progressiva (MP), Distancia média percorrida (DAP), Distancia Curvilínea (DCL), distancia retilínea (DSL), velocidade média de percurso (VAP), Velocidade Curvilínea (VCL), = (VSL), Retilinearidade (STR), Linearidade (LIN), wobble – oscilação (WOB), deslocamento lateral de cabeça (ALH), frequência de batimento cruzado (BCF). Cada amostra foi avaliada em pelo menos 6 à 10 campos escolhidos aleatoriamente, 150 à 200 1117 espermatozoides foram avaliados em cada campo. Foi realizada a análise estatística one-way anova no software Statistix 9.0 (2010). RESULTADOS A motilidade total é um dos principais parâmetros utilizados para avaliar diluentes embora a correlação entre motilidade e viabilidade funcional do espermatozóide em caninos não esteja totalmente estabelecida (IVANOVA-KICHEVA et al., 1997). Como resultado deste estudo encontrou-se diferença estatística significativa na análise da motilidade espermática sendo esta dependente da concentração de serina e do período de conservação sob resfriamento analisado (Tabela 1, Tabela 2, Tabela 3 e Tabela 4). TG0 TG2,5S TG5S TG10S TG20S TG30S TG40 S TG50S TG100 S TG200 S MT 63,3A 59,2AB 61,1A 57,7AB 57,9AB 51,6BC 44,3C 46,7C 27,0D 15,7E MP 57,6A 52,3AB 55,1A 50,1AB 50,5AB 43,0BC 35,5C 35,6C 16,2D 7,8D 29,3AB 31,3A 28,8BC 28,2BC 26,6CD 24,5DE 22,2E 17,6E 15,3F DAP 28,9BC DCL 48,9B 49,1B 54,2A 48,7B 49,2B 47,1B 41,6C 38,8C 30,0D 26,4D DSL 23,4B 24,6AB 26,3A 23,9B 22,5BC 21,1CD 19,6D 17,3E 13,6F 11,7F VAP 63,7B 64,9AB 69,8A 64,3B 62,8BC 58,0CD 54,4CD 48,5E 39,1F 36,1F VCL 107,5B 108,5B 120,4A 108,3B 109,5B 102,7C 91,8C 84,6C 65,9D 61,4D VSL 51,5B 54,4AB 58,4A 53,2B 50,1BC 46,1CD 43,7D 37,8E 30,4F 27,6F STR 0,794BC 0,830A 0,831A 0,816AB 0,785C 0,777CD 0,777CD 0,762CD D E E E 1118 0,792C 0,758E 0,473AB 0,453B C 0,501A 0,491A 0,497A WO B 0,591BC 0,600AB 0,585BC 0,600AB 0,573A D C D C B 0,564D ALH 4,17BCD 4,24BC 4,62A 4,38AB 4,40AB 4,05CD BCF 20,5A 17,7AB 15,9BCD 15,7BCD LIN C 16,2BC D 0,443C 16,3BC 0,485A 0,449C 0,608A 0,588BC B D 0,631A 0,612AB 3,85DE 3,43F 3,06G 3,62EF 15,3BCD 13,3CD 12,1D 15,2BC D 0,500A 0,473AB B C Tabela 1. Efeito da adição diferentes concentrações de serina (0mM, 2,5mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50 mM, 100 mM,200 mM) no diluente TG em amostras resfriadas de sêmen canino sobre parâmetros de motilidade espermática analisado pelo CASA na hora zero (Computer Assisted Sperm Analysis). Letras distintas demonstram diferença estatística nas linhas (P>0.05). Motilidade total (MT, %), Motilidade progressiva (MP, %), Deslocamento lateral de cabeça (ALH, μm), frequência de batimento cruzado (BCF, μm), distância média percorrida (DAP, μm), distância curvilínea (DCL, μm), distância retilínea (DSL, μm), linearidade (LIN, %), retilinearidade (STR, %), velocidade média de percurso (VAP, μm/s), velocidade curvilínea (VCL, μm/s), (VSL, μm/s), wobble – oscilação (WOB, μm/s). Fonte: Dode et al (2022). Tabela 2. Efeito da adição diferentes concentrações de serina (0mM, 2,5mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50 mM, 100 mM,200 mM) no diluente TG em amostras resfriadas de sêmen canino sobre parâmetros de motilidade espermática analisado pelo CASA nas 24 horas (Computer Assisted Sperm Analysis). TG0 TG2,5S TG5S TG10S TG20 S TG30S TG40S TG50 S TG100 S TG200 S MT 64,2A 55,9B 51,7B 40,5C 28,8D 39,7C 29,8D 28,8D 20,6E 18,9E MP 56,7A 47,4B 43,8B 31,4C 20,0D 31,0C 21,3D 17,8DE 10,9E 11,5E DAP 30,6A 28,9AB 27,0B 24,7C 20,9D 23,7C 21,1D 20,9D 19,0D 13,9E DCL 50,7A 49,2AB 45,5BC 43,0CD 35,1F 40,8DE 37,0EF 36,6F 34,5F 20,5G 1119 DSL 25,7A 24,2AB 22,6B 20,1C 17,1DE 19,0CD 16,8E 16,2EF 14,4F 11,6G VAP 67,1A 64,4A 59,9B 54,8C 47,2D 53,5C 46,4D 46,0D 42,7D 33,0E VCL 111,1A 109,1AB 100,6BC 94,8CD 78,2E 91,4D 80,7E 80,2E 76,3E 48,0F VSL 56,4A 53,7AB 50,0B 44,4C 38,8D 43,1C 37,2D 35,8DE 32,7E 27,7F STR 0,831A 0,826AB 0,828A 0,800BC B 0,800BC 0,792CD E 0,763E 0,846A 0,501B 0,491BCD 0,496BC 0,467CD 0,525B 0,479CD 0,474CD C E D E C E E 0,459E 0,460DE 0,632A LIN 0,820A 0,770D WO B 0,600B C 0,589B 0,596C 0,578C 0,632B 0,594C 0,589C 0,589C 0,591C 0,739A ALH 3,96AB 4,25A 3,95AB 3,76BC 3,65BC 3,88B 3,30DE 3,54CD 3,46CD 3,02E BCF 23,4A 17,2B 18,0B 16,7B 13,0CD 15,6BC 17,0B 16,7B 15,2BC 10,8D Letras distintas demonstram diferença estatística nas linhas (P>0.05). Motilidade total (MT, %), Motilidade progressiva (MP, %), Deslocamento lateral de cabeça (ALH, μm), frequência de batimento cruzado (BCF, μm), distância média percorrida (DAP, μm), distância curvilínea (DCL, μm), distância retilínea (DSL, μm), linearidade (LIN, %), retilinearidade (STR, %), velocidade média de percurso (VAP, μm/s), velocidade curvilínea (VCL, μm/s), (VSL, μm/s), wobble – oscilação (WOB, μm/s). Fonte: Dode et al (2022). Tabela 3. Efeito da adição diferentes concentrações de serina (0mM, 2,5mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50 mM, 100 mM,200 mM) no diluente TG em amostras resfriadas de sêmen canino sobre parâmetros de motilidade espermática analisado pelo CASA nas 48 horas (Computer Assisted Sperm Analysis). TG0 TG2,5S TG5S TG10S TG20S TG30S TG40S TG50 S TG100 S TG200 S MT 38,1B 36,7B 47,8A 39,0B 25,3C 28,3C 23,9C 23,7C 10,3D 9,2D MP 29,1B 28,1B 39,7A 29,4B 17,8CD 19,5C 13,2D 11,8D 2,6E 0,9E 24,8ABC 26,8A 25,5AB 22,8CD 20,5E 20,9DE 19,6E 16,8F 13,1E DAP 24,5BC 1120 DCL 42,9AB 43,5AB 45,6A 43,9AB 40,1BC 35,3D 37,0CD 20,1BC 20,4B 22,1A 21,1C 18,5C 16,4D VAP 54,1BC 55,2B 60,1A 56,8AB 50,8C VCL 94,3AB 96,7A 101,9A 97,4A VSL 44,3BC 45,5B 50,1A STR 0,809AB 0,832A LIN 0,466CD 0,465CD WO B 0,572CD DSL E 0,812AB C 36,0C D 29,8E 19,4F 16,5D 14,4E 12,3EF 10,1F 46,4D 46,6D 43,3D 37,5E 35,4E 88,5BC 79,6D 82,0CD 78,7D 65,5E 50,7F 47,0AB 41,3C 37,3D 36,8D 31,8E 27,4E 27,5E 0,819AB 0,808BC 0,782D 0,726 E 0,735E 0,504BC 0,481BC 0,481BC 0,490AB 0,455C D D C 0,453D 0,404 D E D 0,577A 0,583BC 0,589BC D D 0,603B 0,609B 0,716A 0,569DE 0,601BC 0,801BC D 0,576BCD 0,550 E E 0,787C D ALH 3,86CDE 3,94BC 4,17AB 3,91CD 3,92BC 3,78CDE 3,63E 3,65DE 3,68CDE 4,38A BCF 15,7A 17,1A 16,5A 16,8A 16,3A 14,6A 16,9A 16,2A 13,2A 6,2B Letras distintas demonstram diferença estatística nas linhas (P>0.05). Motilidade total (MT, %), Motilidade progressiva (MP, %), Deslocamento lateral de cabeça (ALH, μm), frequência de batimento cruzado (BCF, μm), distância média percorrida (DAP, μm), distância curvilínea (DCL, μm), distância retilínea (DSL, μm), linearidade (LIN, %), retilinearidade (STR, %), velocidade média de percurso (VAP, μm/s), velocidade curvilínea (VCL, μm/s), (VSL, μm/s), wobble – oscilação (WOB, μm/s). Fonte: Dode et al (2022). Tabela 4. Efeito da adição diferentes concentrações de serina (0mM, 2,5mM, 5mM, 10mM, 20mM, 30mM, 40mM, 50 mM, 100 mM,200 mM) no diluente TG em amostras resfriadas de sêmen canino sobre parâmetros de motilidade espermática analisado pelo CASA nas 72 horas (Computer Assisted Sperm Analysis). TG0 TG2,5S TG5S TG10S TG20S TG30S TG40S TG50 S TG100S TG200S MT 23,8B 27,7A 21,6B 15,5C 15,4C 13,8C 16,3C 15,9C 15,8C 11,9C MP 17,2A 18,3A 11,8B 7,3CD 5,0CD 5,3CD 8,2C 6,3CD 5,5CD 1,2D DAP 22,7A 23,4A 22,3A 22,3A 18,6B 17,5B 19,5B 18,8B 17,1B 7,8C 1121 DCL 41,7A 42,1A 41,4A 40,8A 32,2B 30,4BC 35,0B 35,5B 30,7BC 10,2D DSL 17,9A 18,7A 17,8A 17,3A 14,2B 13,1B 14,6B 13,7B 12,6B 6,5C VAP 49,7AB 51,4A 49,0AB 47,5B 41,3C 39,2C 43,2C 40,7C 39,1C 16,1D VCL 90,8A 92,2A 90,4A 86,6A 70,4BC 67,6C 76,9B 76,7BC 68,4BC 21,0D VSL 39,1A 41,0A 39,0A 36,9B 31,6C 29,7C 32,5C 29,6C 29,2C 13,5D STR 0,781A 0,792A 0,788A 0,765AB 0,761A 0,758A 0,745A B B B 0,724B 0,749AB 0,838A LIN 0,432A 0,442A 0,435A 0,426AB 0,464A 0,468A 0,428A B B B 0,389B 0,448AB 0,680A 0,549C 0,554C 0,549C 0,554C D D D D 0,601B 0,610B 0,568C 0,535D 0,588BC 0,808A ALH 3,90A 3,90A 3,68AB 3,35C 3,43BC 3,15C 3,36BC 3,07C 3,63ABC 0,67D BCF 17,4A 16,7A 18,0A 17,8A 13,9BC 14,9ABC 18,2A 16,5AB 19,6A 9,2C WOB Letras distintas demonstram diferença estatística nas linhas (P>0.05). Motilidade total (MT, %), Motilidade progressiva (MP, %), Deslocamento lateral de cabeça (ALH, μm), frequência de batimento cruzado (BCF, μm), distância média percorrida (DAP, μm), distância curvilínea (DCL, μm), distância retilínea (DSL, μm), linearidade (LIN, %), retilinearidade (STR, %), velocidade média de percurso (VAP, μm/s), velocidade curvilínea (VCL, μm/s), (VSL, μm/s), wobble – oscilação (WOB, μm/s). Fonte: Dode et al (2022). A manutenção da motilidade segundo HAY et al. (1997) esta relacionada ao movimento progressivo. Quando analisado pelo sistema Casa logo após a diluição (hora zero, Tabela 1), os resultados para motilidade total e progressiva nos tratamentos TG0 (63,3 e 57,6% respectivamente), TG2,5S (59,2 e 52,3%), TG5S (61,1 e 55,1%), TG10S (57,7 e 50,1%) e TG20S (57,9 e 50,5%) apresentaram resultados superiores aos demais tratamentos. Nos parâmetros DAP, DSL, VAP e VSL analisados resultados obtido nos tratamentos TG2,5S (29,3 e 24,6 μm, 64,9 e 54,4 μm/s respectivamente) e TG5S (31,3 e 26,3 μm, 69,8 e 58,4 μm/s) foram superiores aos demais tratamentos. Após 24 horas (Tabela 2) de resfriamento o tratamento controle apresentou motilidade total e progressiva superiores (64,2 e 56,7%) . Os parâmetros DAP, DCL, DSL, VAP, VCL e VSL nos tratamentos TG0 (30,6, 50,7 e 25,7 μm, 67,1, 111,11 e 56,4 μm/s) e TG2,5S (28,9, 49,2 e 24,2μm, 64,4, 109,1 e 53,7μm/s) foram significativamente superiores. Mesmo após 48 horas (Tabela 3) de resfriamento, os parâmetros motilidade total e progressiva e DAP do tratamento contendo a concentração de serina de 5 mM (47,8 e 1122 39,7%, 22,1μm respectivamente), demonstrou resultados superiores aos demais tratamentos. Nos parâmetros VAP e VSL, obtivemos resultados superiores nos tratamentos TG5S (60,1 e 50,1 μm/s) e TG10S (56,8 e 47,0 μm/s). Após resfriamento de 72 horas (Tabela 4), na avaliação demotilidade total , o tratamento TG2,5S (27,7%)foi superior, já a motilidade progressiva não diferiu nos tratamentos TG2,5S (18,3%) e TG0 (17,2%) sendo esses superiores aos demais. Ja os parâmetros DAP, DCL, DSL e VAP, nos tratamentos TG0 (22,7, 41,7 e 17,9μm e 90,8μm/s), TG2,5S (23,4, 42,1 e 18,7μm e 92,2 μm/s), TG5S (22,3, 41,1 e 17,8μm e 90,4μm/s) e TG10S (22,3, 40,8 e 17,3μm e 86,6μm/s) foram os que apresentados resultados superiores aos demais. Até mesmo após 72h, as amostras apresentaram viabilidade quando foi acrescentado 200mMol de serina ao diluente base Tris-gema (Tabela 4). A Figura 1 representa visualmente as curvas de motilidade total e progressiva das amostras de semen canino resfriado, tratadas com diferentes doses de serina no meio diluente em relação ao controle (dose 0) durante 48h. No tempo 0, a presença do aminoácido até 10mM não altera o percentual de motilidade total e progressiva. Observase a mesma tendência de curva para motilidade total e progressiva no controle e no tratamento contendo 2,5mM de serina e para os tratamentos 5 e 10mM. Apos 24h de tratamento, nas concentrações de 5 e 10mM observa-se um platô com menor perda de viabilidade apontando o efeito protetor de concentrações baixas da serina no meio de diluição durante o processo de refrigeração. Valores de motilidade total e progressiva obtidos no período de refrigeração de 48h no meio diluente contendo 5mM de serina foram superiores aos observados no controle indicando um efeito protetor enquanto que após 72h o meio contendo 2,5mM de serina apresentou motilidade total significativamente superior ao controle, indicando uma ampliação da viabilidade dos espermatozóides. Figura 1.Efeito de diferentes concentrações de Serina (2,5 mM, 5 mM e 10 mM) e o controle (0), em amostras de sêmen canino, resfriados a 4ºC por 72 horas sobre os parâmetros de motilidade espermática progressiva (p) e total (t) analisados no CASA (computer Assisted Sperm Analysis). 1123 70, Motilidade % 52,5 35, 17,5 0, 0 Hora pTG0 tTG0 24 horas pTG2,5S tTG2,5S 48 horas pTG5S tTG5S 72 horas pTG10S tTG10S Fonte: Dode et al (2022). Estudos em diferentes espécies apresentam resultados amplamente variáveis quanto aos aminoácidos a serem utilizados e as concentrações mais adequadas (Tuck et al., 1970; Trimeche et al., 1999;Sexton, 1971; Pena, 2004). CONSIDERAÇÕES FINAIS A adição de serina em concentrações de até 5 mM são promissoras para uso em diluentes de semen canino visando manter os parâmetros de motilidade espermática, sendo o potencial protetor da adição de serina em diluidores de semen canino é dose e tempo dependente. . 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https://openalex.org/W3011393283	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09027829.pdf	English	null	A Home Energy Management System With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling	IEEE access	2,020	cc-by	13,694	HUY TRUONG DINH, (Student Member, IEEE), JAESEOK YUN , DONG MIN KIM , KYU-HAENG LEE , AND DAEHEE KIM , (Member, IEEE) Department of Future Convergence Technology, Soonchunhyang University, Asan 31538, South Korea Corresponding author: Daehee Kim (daeheekim@sch.ac.kr) This work was supported in part by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Minist Industry and Energy (MOTIE), South Korea, under Grant 20184030202130, and in part by the Soonchunhyang University Rese This work was supported in part by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry and Energy (MOTIE), South Korea, under Grant 20184030202130, and in part by the Soonchunhyang University Research Fund. ABSTRACT With the development of new technologies in the ﬁeld of renewable energy and batteries, increasing number of houses have been equipped with renewable energy sources (RES) and energy storage systems (ESS) to reduce home energy cost. These houses usually have home energy management sys- tems (HEMS) to control and schedule every electrical device. Various studies have been conducted on HEMS and optimization algorithms for energy cost and peak-to-average ratio (PAR) reduction. However, none of papers give a sufﬁcient study on the utilization of main grid’s electricity and selling electricity. In this paper, ﬁrstly, we propose a new HEMS architecture with RES and ESS where we take utilization of the electricity of the main grid and electricity selling into account. With the proposed HEMS, we build general mathematical formulas for energy cost and PAR during a day. We then optimize these formulas using both the particle swarm optimization (PSO) and the binary particle swarm optimization (BPSO). Results clearly show that, with our HEMS system, RES and ESS can help to drop home energy cost signiﬁcantly to 19.7%, compared with the results of previous works. By increasing charge/discharge rate of ESS, energy cost can be decreased by 4.3% for 0.6 kW and 8.5% for 0.9 kW. Moreover, by using multi-objective optimization, our system can achieve better PAR with an acceptable energy cost. INDEX TERMS Home energy management systems, electricity selling, renewable energy sources, energy storage systems, day-ahead price, meta-heuristic algorithms. Received February 28, 2020, accepted March 4, 2020, date of publication March 9, 2020, date of current version March 19, 2020. Di i l Obj Id ifi 10 1109/ACCESS 2020 2979189 Received February 28, 2020, accepted March 4, 2020, date of publication March 9, 2020, date of current version March 19, 2020. Digital Object Identifier 10.1109/ACCESS.2020.2979189 under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME 8, 202 This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0 I. INTRODUCTION Moreover, with amazing development of electric mobile devices such as electric vehicles (EV) and revolution of the technologies in RES, consumers are encouraged to participate in electricity market by selling surplus energy. The buyers can be electric cars, other houses and even electricity providers. To achieve the above objectives, a home energy management system (HEMS) is required at the consumer side. HEMS controls and optimizes RES, ESS and home appliances in a most efﬁcient way and also helps consumers to incorporate other DSM activities. • In proposed HEMS, we combine two algorithms, PSO and BPSO, for optimization because of the complexity of the HEMS architecture: BPSO is used for binary variables and PSO is used for continuous variables. • We evaluate the proposed HEMS by performing exten- sive simulations. Firstly, we focus on the minimiza- tion of energy cost using single-objective optimization. Secondly, we apply multi-objective optimization to the HEMS to minimize both energy cost and PAR. Secondly, we apply multi-objective optimization to the HEMS to minimize both energy cost and PAR. • We further analyze our proposed HEMS with the effects of changing the selling price. In addition, the effect of different ESS charge/discharge rate and ESS capacity for reducing the energy cost are also evaluated. The rest of this paper is organized as follows. Section II reviews the related works to our system and our paper’s con- tributions. In Section III, we describe our HEMS architecture in detail. Problem formulation of total energy cost and PAR during a day is built in Section IV. PSO and BPSO algorithms are presented in Section V. In Section VI, simulations and the results are discussed. Finally, Section VII draws the con- clusion of our paper after which some future works are also highlighted. p Various studies have proposed algorithms to optimize the operation of home appliances such as linear program- ming (LP) [6], mixed integer linear programming (MILP) [7], [8], mixed integer nonlinear programming (MINLP) [9], [10], dynamic programming (DP) [11], or convex programming (CP) [12]. However, these techniques have very slow convergence rate with a large number of vari- ables, and in some cases, they are unable to handle a lot of appliances [3], [13]. Meta-heuristic algorithms are employed in HEMS to deal with such shortcomings. I. INTRODUCTION p g proposed HEMS makes the following contributions. p DSM is an important part in the energy management of SG. DSM provides a diversity of functionalities in vari- ous areas such as electricity market control, energy man- agement, infrastructure construction and management of decentralized energy resources [2]. DSM, which includes demand response (DR) and energy optimization, encom- passes a broader range of energy demand management con- cepts. DSM seeks a balance between energy supply and demand on the both side of utilities and consumers whereas DR focuses on the consumer side only. The DR is used for the programs designed to help end-users to reduce short- term energy demand in response to a price signal from the electricity hourly market, or a trigger initiated by the elec- tricity grid operator [3]. DR seeks to adjust the demand for power, rather than the supply. However, it is examined in the literature that researchers considered the DSM and DR are interchangeable [4]. • With the integration of RES and ESS, we propose a novel HEMS architecture to reduce energy cost and PAR. In our HEMS, utilizing main grid and electricity selling are emphasized and described as follows: Our ESS can be used to store electricity from main grid at low price time as well as electricity from RES. This electricity is reused for home load at high price time. Moreover, our HEMS allows prosumers to sell surplus electricity at appropriate time slots. • With the proposed HEMS architecture, we build general mathematical formulas where we consider utilization of main grid and electricity selling as well as the sell- ing price. Based on these formulas, our HEMS gives detailed schedules for each electrical device during a day. Furthermore, the amount of electricity utilized from main grid and the amount of electricity sold to the out- side are also determined at each time slot. Usually, the main objective of DSM and DR functions in SG is to encourage the consumers to utilize their RES (local generators) for their load, especially at peak hours, whereby decreasing the dependence on electricity providers. The sec- ond objective is to encourage consumers to shift home load: moving the time of energy use from peak hours to off-peak hours [2], [5]. These objectives also help the consumers to reduce their electricity cost and energy consumption in peak hours. I. INTRODUCTION The most widely used meta-heuristic algorithms are particle swarm optimization (PSO), binary particle swarm optimiza- tion (BPSO), genetic algorithm (GA), wind-driven optimiza- tion (WDO), bacterial foraging optimization (BFO) and Jaya algorithm [3], [13], [14], [15]. I. INTRODUCTION For the purpose of utilization, smart grid (SG) has been introduced as a replacement of the existing power grid which does not support consumer’s new requirements. The SG utilizes the latest communication technologies to improve the traditional electrical power system. With integration of RES and ESS, SG helps decrease the emission of carbon particulate and greenhouse gases as well as climate change mitigation (CCM) [1]. Two key integral parts of SG which make it better than traditional grids are advanced metering infrastructure (AMI) and demand side management (DSM). In recent decades, the rate of global warming and climate change have been more severe, causing world extreme events such as hemispherical sea ice melting, serious ﬂood, strong hurricane, and so on. One of main causes of global warming is carbon dioxide emissions from the consumption of fossil fuel to meet daily energy demand. To mitigate this prob- lem, researches have been conducted in two different ways: ﬁnding more renewable energy resources (RES) to replace fossil fuel and utilizing energy in a most efﬁcient way with the integration of RES and energy storage systems (ESS). The main functions of AMI are energy measurement and information collection. It comprises of smart meters and Information and Communication Technology (ICT). The ICT enables SG to keep the consumers updated about varying The associate editor coordinating the review of this manuscript and approving it for publication was Alexander Micallef . 49436 VOLUME 8, 2020 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling electricity prices, or events/failures due to devices or disas- ters. It also sends information of energy consumption mea- sured by smart meter to electricity operators to enable the operators to monitor and analyze real-time data and make real-time decisions about activities of power networks. electricity prices, or events/failures due to devices or disas- ters. It also sends information of energy consumption mea- sured by smart meter to electricity operators to enable the operators to monitor and analyze real-time data and make real-time decisions about activities of power networks. Based on the above background, we propose a new HEMS with integration of RES and ESS. Our HEMS is connected with electricity operator through AMI. Electricity operator sends useful forecast information to our HEMS by using AMI such as pricing information and solar irradiance. The proposed HEMS makes the following contributions. II. RELATED WORK AND MOTIVATION Their simu- lation results showed that with the support of their system, energy consumption was decreased from 1360.9 kWh to 820.2 kWh. Hence, energy cost of their system was reduced from $62.91 to $40.37. However, the utilization of main grid and PAR were not considered. In [3], an optimized home energy management sys- tem (OHEMS) with integrated RES and storage resource to optimize the energy cost and PAR was proposed. The tariff in their system was a day-ahead pricing. They applied many heuristic algorithms into OHEMS and compared results of these algorithms. These heuristic algorithms are genetic algorithm (GA), binary particle swarm optimization (BPSO), wind driven optimization (WDO), bacterial foraging opti- mization (BFO) and hybrid GA-PSO (HGPO). Their study shows that HGPO gives the lowest total energy cost and BFO gives the lowest PAR among these algorithms. However, the user comfort (UC) was not discussed. In this work, authors gave a ﬁxed plan for the operation of RES and ESS. ESS is only used to store 30% of RES energy at day time and discharged at high price time slot at night time. 70% of RES energy is used for home load. With this ﬁxed plan, their OHEMS cannot utilize the electricity of the main grid at time which has low price. Moreover, their OHEMS does not support consumers to sell electricity. In [13], HEMS with different heuristic algorithms and dif- ferent tariffs was studied. Their objectives were to minimize energy cost, PAR and maximize user comfort. The user com- fort in their papers is waiting time of user. The tariffs applied in their system were real-time electricity pricing (RTEP) and critical peak pricing (CPP). Four heuristic algorithms applied in their system were wind driven optimization (WDO), har- mony search algorithm (HSA), genetic algorithm (GA) and GHSA which combines the attributes of GA and HSA. They also considered their HEMS in two cases single home (SH) and multiple homes (MH). Their simulation results showed that GHSA outperformed the other algorithms in terms of objectives. However, in their HEMS, the integration of RES and ESS into residential side was ignored. p g In [16], an ontology-driven multi-agent based energy man- agement system (MAS) was proposed. This system was used to monitor and optimally control a micro-grid system with integrated homes or buildings (residential micro-grid) with various RES. II. RELATED WORK AND MOTIVATION II. RELATED WORK AND MOTIVATION Recently, various studies have been conducted on HEMS with various optimization algorithms. The common targets of these studies were to minimize energy cost and PAR. Beside the two objectives, some papers also consider user comfort (UC) such as waiting time, thermal comfort, air quality and so on. In [9], a home automation/energy management sys- tem (HAEMS) with integrated ESS was presented. 49437 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling The objective of this paper was to optimize a mixed objective function which includes energy cost, user’s convenience and thermal comfort. The tariff used in their system was RTP pricing. In this paper, they used General Algebraic Modeling System (GAMS) software with Cplex/Dicopt solvers as the main optimization engine. They considered their HAEMS in three scenarios: naive, normal, and smart. The simulation results showed that in smart scenario, their HAEMS has improved the mixed objective function up to 55% and 25% with respect to the naive and normal scenarios in a hot weather condition and up to 63% and 38% in a cold weather condition. However, in this paper, RES was not included and PAR was not considered. In [10], authors proposed a residential smart energy management system (RSEMS) with integrated ESS and RES. A hybrid objective function was built to optimize the energy cost, user’s satisfaction and thermal comfort simultaneously. They used MATLAB and GAMS for optimization tasks. Their RSEMS was compared with conventional EMS in two scenarios: hot summer day and cold winter day. Their RSEMS helped to improve hybrid objective function up to 29% and 33% in hot and cold weather conditions respectively. However, PAR was not considered in this paper. In both [9] and [10], utilizing main grid’s electricity at low price time and electricity selling activities were mentioned but they were not emphasized. Authors did not give a detailed schedule for selling operation at each time slot. Moreover, selling price was not considered. Their works can not be applied in case selling price is smaller than the price of main grid. adopted to model power trading between these users. At each time slot, ECC was re-run to turn on or off home appli- ances and users could re-choose selling price and amount of trading energy based on Nash equilibrium. II. RELATED WORK AND MOTIVATION However, their system does not have AMI, hence they did not receive information from electricity operators. In their system, they forecast daily load in general and home appliances were not described. They did not give a detailed schedule for each device at home. Hence, this system cannot be applied to DSM. In addition, authors only consider minimizing energy cost without taking PAR into account. In [18] and [19], straightforward solution of HEMS was proposed with direct current (DC) power management. In their HEMS, smart DC sockets with load shedding algo- rithm were used to control home devices with priority. How- ever, home devices were turned on or off by smart sockets only according to the threshold of energy consumption at each sampling period, and time constraints of home devices were not considered. FIGURE 1. HEMS architecture. electricity providers to consumers. SM works as a commu- nication gateway between our house and providers. SM is responsible for reading, processing and sending energy usage data from our house to the providers via AMI. Moreover, AMI is also used to transmit useful grid information from electricity providers to consumers. These kinds of informa- tion may be the price information, forecast temperature, solar irradiance, and wind speed. In [6], an OEMS for reduction of energy cost was studied. In this work, plug-in hybrid electric vehicle (PREV) batteries and ESS were used to collect electricity and determine the optimization values. The PREV provides electricity for ESS when demand is low and ESS is discharged for home when demand is high. They used linear programming to solve the optimization problem. However, RES was not considered in this model. The MC is the heart of our HEMS. Main task of MC is to control all home appliances including a PV system and ESS. MC is able to turn on or off every device in our system based on schedule calculated through our optimization algorithms. A smart scheduler (SS) that performs optimization algorithms is installed inside MC. At the beginning of the day, the useful information is received from electricity provider via AMI. SS is then run to create an optimized schedule for every device during a day. MC can control all appliances following this schedule to achieve the minimization of energy cost and PAR. In [20], an energy management system with load fore- casting based on Kalman Filter was demonstrated. II. RELATED WORK AND MOTIVATION In their system, a PV system, ESS and critical peak pricing (CPP) were used. ESS was used to store excess PV power and provide the charged power as needed. Kalman Filter was used to forecast the home load for the next day. Their system need to be trained to construct load forecasting model. How- ever, their system did not have AMI to receive information from electricity operators. The DSM and DR activities were also not considered. In addition, their system did not give a detailed schedule for each home appliance. There are many kinds of RES such as wind turbines, PV systems, fuel cells. In our system, we use a PV system because of its easy installation and cheap price. A DC/AC inverter is used to convert the DC current from the PV system into AC current. The brief comparison of research works on HEMS is listed in Table 1 where a HEMS supporting selling operation allows prosumers to change selling price and give a detailed amount of energy sold to the outside. On the other hand, selling capa- bility means that a HEMS is considered to sell electricity to the outside but it does not support to change selling price and does not give a schedule for selling. Motivated by the above literature works, we suggest a novel HEMS with integration of RES and ESS utilizing main grid and electricity selling whose objective is to minimize both energy cost and PAR. To utilize the electricity of the main grid, we use ESS to store electricity from the main grid at low price time and provide electricity for our home appliances at high price time. ESS also helps us exploit the PV system efﬁciently. The energy from a PV system is able to be stored in ESS at any time slot and reused in different time slot. Furthermore, our system supports selling energy to the outsiders such as smart electric vehicles, other houses, or even main grid. We assume that our MC can use AMI to transmit selling electricity. II. RELATED WORK AND MOTIVATION At homes, different agents including EMS were implemented to cooperate with each other to reach an optimal operating strategy. Moreover, this system also had useful agents such as central coordinator agent (CCA) which is responsible for collecting and sharing useful information, and battery bank agent (BBA) to compensate any real-time power imbalances within the residential micro-grid economically. The BBA was also a device which stores the surplus of energy and provides energy back to micro-grid. The BBA was able to sell or buy energy from utility. Authors tested their system in three scenarios: naive, normal, and smart with different time frames (weekdays and weekend), different climate (hot and cold), pricing schemes (RTP, TOU, and ﬂat rate). Through a number of simulations, they demonstrated that the proposed MAS had the capability to reduce system’s operation cost and to ensure user’s needs under different weather conditions, time frames, and pricing schemes. However, MAS focused on the whole residential micro-grid, not for a single home. Muhammad Awais in [17] presented home energy management (HEM) with three heuristic algorithms: bacte- rial foraging optimization algorithm (BFOA), ﬂower pollina- tion algorithm (FPA) and hybrid bacterial ﬂower pollination algorithm (HBFPA). They test the proposed scheme in a sin- gle home and in smart community involving multiple house- holds. Their targets were to minimize energy cost and PAR with affordable users’ waiting time. The proposed HBFPA shows efﬁcacy for energy cost and for reduction of PAR with reasonable user waiting time. However, they did not consider RES and ESS in their HEM. In [11], authors proposed an energy management system for a group of homes with integrated ESS and RES to opti- mize the energy cost. At each home, an energy consumption controller (ECC) with dynamic programming was suggested for load scheduling and a game theoretic approach was In [8], an optimization and energy management in the smart home was proposed. Energy consumption was opti- mized with the integration of ESS, electric vehicle, and two 49438 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling FIGURE 1. HEMS architecture. kinds of RES: solar energy and wind energy. The selling energy was also considered. The system was simulated in three scenarios: 1 day, 4 days and 7 days. MILP and heuristic algorithms were compared in the three scenarios. III. HEMS ARCHITECTURE Fig. 1 shows the main elements of the proposed HEMS. We assume that every consumer is equipped with AMI, a main controller (MC), ESS and a PV system as RES. VOLUME 8, 2020 A. ELECTRICITY FLOWS (4) Fig. 2 shows all electricity ﬂows in our system. In our model, RES can be used to provide electricity for appliances in a house and store electricity in the ESS at any time slot in any quantity. The ESS can be used to provide electricity for appliances and sell electricity at any time slot in any quantity. If the electricity from RES and ESS is not enough for home devices, the electricity of the main grid is needed. Moreover, electricity from the main grid can be stored in ESS in the low price time slot and would be used in the high price time slot. (5) C. ENERGY STORAGE SYSTEM The main role of ESS is to exploit the PV system and the electricity of the main grid more efﬁciently. Our ESS is able to store the energy of the main grid or RES in a time slot with low price and provide to home load in a time slot with high price. The parameters of our ESS used in this paper are shown in Table 2. IV. PROBLEM FORMULATION to 12 P.M.. We also divide a day into T = 24 time slots and the duration of each time slot is 1t = 1h. to 12 P.M.. We also divide a day into T = 24 time slots and the duration of each time slot is 1t = 1h. 0 ≤Eload RES (t) ≤GHI(τ) · S · ηRES · 1t ∀t 1 ≤t ≤T (4) 0 ≤Echarge RES (t) ≤GHI(τ) · S · ηRES · 1t ∀t 1 ≤t ≤T (5) IV. PROBLEM FORMULATION In time slot t with time slot’s duration 1t, our PV syst generates an electrical energy ERES(t) as follows. ERES(t) = PRES(τ) · 1t ∀t 1 ≤t ≤T where τ is the real time in time slot t. As shown in Fig. 2, this energy would be used for ho load and ESS charging. Thus, we have the following equatio ERES(t) = Eload RES (t) + Echarge RES (t) ∀t 1 ≤t ≤T where Eload RES (t) is an energy quantity used for home load time slot t. Echarge RES (t) is an energy quantity used to char ESS in time slot t. From (1), (2), and (3), we have following constraints variables Eload RES (t) and Echarge RES (t). 0 ≤Eload RES (t) ≤GHI(τ) · S · ηRES · 1t ∀t 1 ≤t ≤T 0 ≤Echarge RES (t) ≤GHI(τ) · S · ηRES · 1t ∀t 1 ≤t ≤T C. ENERGY STORAGE SYSTEM The main role of ESS is to exploit the PV system and electricity of the main grid more efﬁciently. Our ESS is ab to store the energy of the main grid or RES in a time slot w TABLE 1. A comparison of HEMS: State of the art. TABLE 1. A comparison of HEMS: State of the art. FIGURE 2. Electricity flows in our HEMS. panels and ηRES is the solar conversion efﬁciency of the PV system. In time slot t with time slot’s duration 1t, our PV system generates an electrical energy ERES(t) as follows. ERES(t) = PRES(τ) · 1t ∀t 1 ≤t ≤T (2) (2) where τ is the real time in time slot t. where τ is the real time in time slot t. As shown in Fig. 2, this energy would be used for home load and ESS charging. Thus, we have the following equation. ERES(t) = Eload RES (t) + Echarge RES (t) ∀t 1 ≤t ≤T (3) (3) where Eload RES (t) is an energy quantity used for home load in time slot t. Echarge RES (t) is an energy quantity used to charge ESS in time slot t. FIGURE 2. Electricity flows in our HEMS. From (1), (2), and (3), we have following constraints for variables Eload RES (t) and Echarge RES (t). to 12 P.M.. We also divide a day into T = 24 time slots and the duration of each time slot is 1t = 1h. IV. PROBLEM FORMULATION To optimize electricity bill and PAR, we deﬁne mathematical models and constraints of all elements in our HEMS. In this section, we build mathematical formulas of RES, ESS, appli- ances and our cost function during a day time from 0 A.M. In general, AMI refers to collection of systems that include smart meter (SM), advanced communications and data man- agement systems [13]. AMI works as a backbone from 49439 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling TABLE 1. A comparison of HEMS: State of the art. FIGURE 2. Electricity flows in our HEMS. to 12 P.M.. We also divide a day into T = 24 time slots and the duration of each time slot is 1t = 1h. A. ELECTRICITY FLOWS Fig. 2 shows all electricity ﬂows in our system. In our model, RES can be used to provide electricity for appliances in a panels and ηRES is the s system. In time slot t with tim generates an electrical e ERES(t) = PRE where τ is the real time As shown in Fig. 2, t load and ESS charging. T ERES(t) = Eload RES (t where Eload RES (t) is an ene time slot t. Echarge RES (t) is ESS in time slot t. From (1), (2), and (3 variables Eload RES (t) and E 0 ≤Eload RES (t) ≤GHI( 0 ≤Echarge RES (t) ≤GHI TABLE 1. A comparison of HEMS: State of the art. FIGURE 2. Electricity flows in our HEMS. to 12 P.M.. We also divide a day into T = 24 time slots and the duration of each time slot is 1t = 1h. A. ELECTRICITY FLOWS Fig. 2 shows all electricity ﬂows in our system. In our model, RES can be used to provide electricity for appliances in a house and store electricity in the ESS at any time slot in any quantity. The ESS can be used to provide electricity for appliances and sell electricity at any time slot in any quantity. If the electricity from RES and ESS is not enough for home devices, the electricity of the main grid is needed. Moreover, electricity from the main grid can be stored in ESS in the low panels and ηRES is the solar conversion efﬁciency of the P system. D. HOME APPLIANCES In our system, we suppose that there are two different sets of appliances: shiftable appliances M and non-shiftable appli- ances N. The set of shiftable devices M = {a1, a2, a3, ..., am} includes the devices which can operate at any time slots whereby we can move the operation time of these devices to low price slots to save costs. The set of non-shiftable devices N = {b1, b2, b3, ..., bn} have a ﬁxed operation time slots deﬁned by users. In a time slot t, the energy consumption of total appliances, Eappliances total (t), in a house is the sum of the energy consumption of shiftable set M, EM(t), and non- shiftable set N, EN(t), which are given in (18), (19), (20), with ∀bi ∈N, ∀ai ∈M, ∀t 1 ≤t ≤T. modeESS(t) = ( 1 if ESS is charged. 0 if ESS is discharged. (8) (8) Assuming that ELevel ESS (t) is energy level of ESS after time slot t where ∀t 1 ≤t ≤T, we have the following formula. ELevel ESS (t) = ELevel ESS (t −1) + ECharge ESS (t) · ηESS Eappliances total (t) = EN(t) + EM(t) (18) EN(t) = n X i=1 Powerrate(bi) × O(bi, t) × 1t (19) EM(t) = m X i=1 Powerrate(ai) × O(ai, t) × 1t (20) −EDischarge ESS (t)/ηESS (9) (18) (9) where ηESS is ESS efﬁciency. It is worth noting that ηESS must be used in (9) because some energy is lost when charging or discharging an ESS, which is called round trip efﬁciency. When using ESS, we must satisfy the following constraints. (19) When using ESS, we must satisfy the following constraints. (20) • The charge/discharge rate of ESS cannot exceed the Chrate/Dhrate. It means that we are only able to put in or draw certain energy quantity in a time slot t with duration 1t. where Powerrate(ai) and Powerrate(bi) refer to the power rat- ing of devices ai, bi which is given by producers. O(ai, t) and O(bi, t) are binary variables which show the status of devices ai and bi in a time slot t. • The energy level of ESS must be between ELmin and ELmax. • The energy level of ESS must be between ELmin and ELmax. B. RENEWABLE ENERGY SOURCE Eload ESS (t) is an energy quantity used for home load in a time slot t. Eselling ESS (t) is an energy quantity used to sell to the outside in a time slot t. Echarge RES (t) is an energy quantity stored in ESS from RES in a time slot t. Echarge MG (t) is an energy quantity stored in ESS from the main grid in a time slot t. Because ESS is only able to be either charged or discharged in a time slot, modeESS(t) is a binary variable which shows the status of ESS in slot t. B. RENEWABLE ENERGY SOURCE As shown in Fig. 1, our HEMS is equipped with a PV system as RES. According to [21] and [22], we use the following equation to calculate the output power PRES of PV system in kW at time τ. In a general case, ESS in our HEMS has two functions: a source to provide energy for home load and sell surplus energy to the outside, and a sink to store energy from RES and the main grid. Hence, with ∀t 1 ≤t ≤T, we have the following formulas. PRES(τ) = GHI(τ) · S · ηRES ∀τ 0 ≤τ ≤24 (1) (1) EDischarge ESS (t) = Eload ESS (t) + Eselling ESS (t) · 1 −modeESS(t) (6) EDischarge ESS (t) = Eload ESS (t) + Eselling ESS (t) · 1 −modeESS(t) where GHI is the global horizontal irradiation (kW/m2) at the location of solar panels. S is the total area (m2) of solar (6) 49440 VOLUME 8, 2020 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling TABLE 2. The parameters of an ESS [21]. ECharge ESS (t) = Echarge RES (t) + Echarge MG (t) · modeESS(t) (7) 0 ≤Echarge MG (t) ≤Chrate · 1t (16) TABLE 2. The parameters of an ESS [21]. (16) Since we only consider our system during a day (no net accumulation for next day), energy level must be returned to the initial energy level at the end of the day. Thus, we have this constraint. ELevel ESS (T) = EL0 (17) (17) We assume that all energy to be sold come from ESS. If we want to sell energy generated from RES, it should be stored in ESS before selling. Note that the variable Echarge RES (t) has two constraints in (5) and (15). If our RES generates more energy than the sum of the energy needed by home appliances and the energy is able to stored in ESS in a time slot, the remain energy of RES will be wasted. (7) where EDischarge ESS (t) refers to an energy quantity which is drawn from ESS in a time slot t. ECharge ESS (t) refers to an energy quantity stored in the ESS in a time slot t. E. LOAD DEMAND AND COST FUNCTION Thus, the objective function of our system becomes From (26), we have ELD(t) = EN(t) + EM(t) + Echarge MG (t)−Eload RES (t)−Eload ESS (t). min T X t=1 EN(t) + m X i=1 Powerrate(ai) × O(ai, t) × 1t + Echarge MG (t) −Eload RES (t) −Eload ESS (t) −α × Eselling ESS (t) × PMG(t) . (35) (29) (29) In addition, we sell amount of energy, Eselling ESS (t), to the out- side in a time slot t. Hence, the energy cost to be paid in a time slot t, EC(t), is (35) EC(t) = ELD(t) × PMG(t) −Eselling ESS (t) × Psell(t). (30) (30) D. HOME APPLIANCES From (29), we have the following formula. (25) Cday = T X t=1 EN(t) + EM(t) + Echarge MG (t) −Eload RES (t) −Eload ESS (t) × PMG(t) −Eselling ESS (t) × Psell(t) (32) From (18), we have −Eload ESS (t) × PMG(t) −Eselling ESS (t) × Psell(t) (32) Eload MG (t) = EN(t) + EM(t) −Eload RES (t) −Eload ESS (t). (26) (32) (26) Because Eload MG (t) ≥0 and we assume that the main grid always provide enough electricity for the requirement of our home load. Thus, we have the following constraint. Since our objective is to minimize the total energy cost during a day, objective function is deﬁned as min(Cday) min(Cday) min(Cday) = min T X t=1 EN(t) + EM(t) + Echarge MG (t) −Eload RES (t) −Eload ESS (t) × PMG(t) −Eselling ESS (t) × Psell(t) . (33) 0 ≤Eload RES (t) + Eload ESS (t) ≤EN(t) + EM(t) = Eappliances total (t) (27) 0 ≤Eload RES (t) + Eload ESS (t) ≤EN(t) + EM(t) = Eappliances total (t) (27) E. LOAD DEMAND AND COST FUNCTION (33) In this subsection, we build a formula of energy from the main grid called load demand in each time slot. We get the total energy cost for a day using the load demand and the prices of the main grid. According to [23], there are many kinds of electricity tariffs such as Time-of-Use pricing (ToU), Real- Time Pricing (RTP), Critical Peak Pricing (CPP) and so on. ToU and RTP are commonly used tariffs in most HEMSs. In this paper, we use Day-Ahead Pricing (DAP), a kind of RTP where the price of electricity changes on the hourly basis and remains constant in an hour. Customers are typically notiﬁed of DAP prices on a day-ahead basis. Combining with (20), we have the objective function of our system as min T X t=1 EN(t) + m X i=1 Powerrate(ai) × O(ai, t) × 1t + Echarge MG (t) −Eload RES (t) −Eload ESS (t) × PMG(t) −Eselling ESS (t) × Psell(t) . (34) min T X t=1 EN(t) + m X i=1 Powerrate(ai) × O(ai, t) × 1t + Echarge MG (t) −Eload RES (t) −Eload ESS (t) × PMG(t) −Eselling ESS (t) × Psell(t) . (34) (34) We assume that the energy from RES and ESS is compli- mentary, whereby in a time slot t, load demand needed from main grid, ELD(t), includes Eload MG (t) and Echarge MG (t) as shown in Fig. 2. With ∀t 1 ≤t ≤T, we have the following formula. In (34), Powerrate(ai), EN(t), and PriceMG(t) are ﬁxed values we already know. O(ai, t) are binary variables. Echarge MG (t), Eload RES (t), Eload ESS (t), Eselling ESS (t) are variables which must satisfy all constraints: (3), (4), (5), (10), (11), (12), (13), (14), (11), (16), (17), (27). In (34), Powerrate(ai), EN(t), and PriceMG(t) are ﬁxed values we already know. O(ai, t) are binary variables. Echarge MG (t), Eload RES (t), Eload ESS (t), Eselling ESS (t) are variables which must satisfy all constraints: (3), (4), (5), (10), (11), (12), (13), (14), (11), (16), (17), (27). ELD(t) = Eload MG (t) + Echarge MG (t) (28) (28) Usually, the price of the main grid is higher than the selling price. We assume that Psell(t) = α ×PMG(t) with 0 < α ≤1. D. HOME APPLIANCES O(ai, t) = ( 1 if shiftable device ai is ON 0 if shiftable device ai is OFF (21) O(bi, t) = ( 1 if non-shiftable device bi is ON 0 if non-shiftable device bi is OFF (22) From above constraints, with ∀t 1 ≤t ≤T, we have the following constraints. (21) 0 ≤EDischarge ESS (t) = Eload ESS (t) + Eselling ESS (t) ≤Dhrate · 1t O(bi, t) = ( 1 if non-shiftable device bi is ON 0 if non-shiftable device bi is OFF (22) (10) (22) 0 ≤ECharge ESS (t) = Echarge RES (t) + Echarge MG (t) ≤Chrate · 1t 0 ≤ECharge ESS (t) = Echarge RES (t) + Echarge MG (t) ≤Chrate · 1t Since O(bi, t) has a ﬁxed value, EN(t) also has ﬁxed value. In a whole day of T = 24 hours, the total energy consumption of all appliances in our system is given by (11) ELmin ≤ELevel ESS (t) ≤ELmax 0 ≤Eload ESS (t) ≤Dhrate · 1t 0 ≤Eselling ESS (t) ≤Dhrate · 1t 0 ≤Echarge RES (t) ≤Chrate · 1t T X t=1 Eappliances total (t) = T X t=1 EN(t) + T X t=1 EM(t). (23) (23) When a shiftable device is moved to low price slots, the energy demand of this device is not changed because the VOLUME 8, 2020 49441 VOLUME 8, 2020 49441 VOLUME 8, 2020 49441 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling for energy from the main grid during a day T = 24 hours, Cday, is operation time of each shiftable device does not change and it is not interrupted during operation. To provide enough energy for home appliances, we use 3 different sources as shown in Fig. 2: energy from RES Eload RES , ESS Eload ESS , and the main grid Eload MG in a time slot t. Hence, we have the following formula with ∀t 1 ≤t ≤T. Cday = T X t=1 EC(t) = T X t=1 ELD(t)×PMG(t) −Eselling ESS (t)×Psell(t) . (31) Eappliances total (t) = Eload RES (t) + Eload ESS (t) + Eload MG (t) (24) ⇒Eload MG (t)=Eappliances total (t)−Eload RES (t)−Eload ESS (t) Eappliances total (t) = Eload RES (t) + Eload ESS (t) + Eload MG (t) (24) (24) From (29), we have the following formula. F. PEAK-TO-AVERAGE RATIO where PMG(t) is the electricity price of the main grid in the time slot t. This value is determined by the electrical provider. Psell(t) is the price of selling energy in the time slot t. This value is decided by users. From (30), total cost we must pay PAR is a ratio of the peak load demand and the average of total load demand over a day, from t = 1 to t = 24. PAR tells the energy behavior of our system and it is directly related to the operation of the electricity main grid. The power supply 49442 49442 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling FIGURE 3. A flowchart of PSO algorithm. FIGURE 3. A flowchart of PSO algorithm. companies always want to keep customers’ PAR low. In our system, it is calculated as follows. PAR = max(ELD(t)) 1 T T X t=1 ELD(t) (36) (36) where ELD(t) is calculated by (29). where ELD(t) is calculated by (29). TABLE 3. PSO and BPSO parameters. TABLE 3. PSO and BPSO parameters. TABLE 3. PSO and BPSO parameters. vi(t + 1) = ω · vi(t) + Cl · rl · (lbi(t) −xi(t)) + Cg · rg · (gb(t) −xi(t)) (37) xi(t + 1) = xi(t) + vi(t + 1) (38) (37) (38) (38) where ω is the inertia weight and is a constant, vi(t) is the velocity of particle at iteration t, Cl is the acceleration coef- ﬁcients for personal best and is a constant, rl is the random number distributed from 0 to 1 for personal best, lbi(t) is the personal best position of the particle at iteration t, xi(t) is the position of the particle at iteration t, Cg is the acceleration coefﬁcients for global best and is a constant, rg is the random number distributed from 0 to 1 for global best, and gb(t) is the global best position at iteration t. After running a number of iterations, all particles will move to a best position (best solution) of the problem. where rand() is function which generate a pseudo-random number in range [0.0, 1.0]. Fig. 3 shows the working ﬂow of our PSO algorithm. The PSO and BPSO parameters used in our simulation are shown in Table 3. V. PARTICLE SWARM OPTIMIZATION ALGORITHM The particle swarm optimization (PSO) is an evolution- ary computation algorithm which simulates the behavior of organisms [24]. The PSO algorithm is usually used to solve continuous optimization problems. At the beginning of PSO algorithm, a population of particles is created and randomly placed at the search space of the problem to be optimized. At each iteration, each particle moves to a different position inside the search space to ﬁnd an optimal solution. A new position is calculated using the current position and velocity. Generally, the new position xi(t + 1) and velocity vi(t + 1) of particle i at iteration t + 1 is calculated by the following formula. FIGURE 3. A flowchart of PSO algorithm. FIGURE 3. A flowchart of PSO algorithm. VI. SIMULATIONS AND DISCUSSIONS Description of the appliances. FIGURE 4. Hourly prices according to DAP signal. TABLE 5. The input parameters of our ESS in the simulation. FIGURE 5. Solar irradiance to compute PRES(τ). TABLE 4. Description of the appliances. TABLE 4. Description of the appliances. TABLE 4. Description of the appliances. TABLE 4. Description of the appliances. FIGURE 4. Hourly prices according to DAP signal. TABLE 5. The input parameters of our ESS in the simulation. FIGURE 4. Hourly prices according to DAP signal. TABLE 5. The input parameters of our ESS in the simulation. FIGURE 5. Solar irradiance to compute PRES(τ). FIGURE 5. Solar irradiance to compute PRES(τ). FIGURE 4. Hourly prices according to DAP signal. ABLE 5. The input parameters of our ESS in the simulation. signal, solar irradiance and RES as shown in Table 4, Fig. 4, Fig. 5 and Fig. 6, respectively. There are 11 appliances that were divided into two categories: shiftable and non-shiftable. The shiftable appliances are devices whose operating time can be shifted to low price time slots whereas operating time of non-shiftable devices cannot be changed. All the appliances cannot be interrupted during operation. Table 4 shows the power rating and the length of operation time of all appliances. For the RES in our system, we used an electricity generation by PV system modeled in (1). Our RES mainly depends on energy conversion efﬁciency of the solar generator, the area of solar cells, solar irradiation. Our RES is conﬁgured to generate the same amount of energy as in [3]. FIGURE 6. Hourly RES energy generated by the PV system. Section IV, the energy from RES is not only used for home appliances but also to charge ESS with any quantity and at any time slot. An energy quantity of ESS also can be used for selling to the outside at any time slot. In our system, we use ESS with the same conﬁguration as in [3]. The parameters of our ESS are shown in Table 5. In [3], authors proposed a ﬁxed plan for RES and ESS. In their work, 30% of energy from RES in each time slot is used for the charging of ESS, and the remaining energy is used for home load. The ESS is charged only from the PV system in the day time. VI. SIMULATIONS AND DISCUSSIONS signal, solar irradiance and RES as shown in Table 4, Fig. 4, Fig. 5 and Fig. 6, respectively. There are 11 appliances that were divided into two categories: shiftable and non-shiftable. The shiftable appliances are devices whose operating time can be shifted to low price time slots whereas operating time of non-shiftable devices cannot be changed. All the appliances cannot be interrupted during operation. Table 4 shows the power rating and the length of operation time of all appliances. For the RES in our system, we used an electricity generation by PV system modeled in (1). Our RES mainly depends on energy conversion efﬁciency of the solar generator, the area of solar cells, solar irradiation. Our RES is conﬁgured to generate the same amount of energy as in [3]. In our system, we use ESS with the same conﬁguration as in [3]. The parameters of our ESS are shown in Table 5. In [3], authors proposed a ﬁxed plan for RES and ESS. In their work, 30% of energy from RES in each time slot is used for the charging of ESS, and the remaining energy is used for home load. The ESS is charged only from the PV system in the day time. The energy in ESS is only used for home load at high price time slot from t20 to t24. In this paper, we propose a fully ﬂexible general plan for RES and ESS. As described in FIGURE 5. Solar irradiance to compute PRES(τ). FIGURE 6. Hourly RES energy generated by the PV system. Section IV, the energy from RES is not only used for home appliances but also to charge ESS with any quantity and at any time slot. An energy quantity of ESS also can be used for selling to the outside at any time slot. A. CASE 1: SINGLE-OBJECTIVE OPTIMIZATION In this case, we focus on minimizing total energy cost of our system during a day. We ﬁrstly assume that Psell(t) = PMG(t)∀t which means α = 1. FIGURE 5. Solar irradiance to compute PRES(τ). FIGURE 6. Hourly RES energy generated by the PV system. Section IV, the energy from RES is not only used for hom appliances but also to charge ESS with any quantity and a any time slot. An energy quantity of ESS also can be used fo selling to the outside at any time slot. TABLE 4. VI. SIMULATIONS AND DISCUSSIONS The energy in ESS is only used for home load at high price time slot from t20 to t24. In this paper, we propose a fully ﬂexible general plan for RES and ESS. As described in VI. SIMULATIONS AND DISCUSSIONS In this section, the results of our simulation are presented. We simulate the hourly energy use of the set of household appliances during a day. We divide a day into 24 time slots where time slot 1 begins from 0 A.M. to 1 A.M., time slot 2 from 1 A.M. to 2 A.M. and so on. Our HEMS was evaluated in two cases: (I) single-objective optimization and (II) multi- objective optimization. In the ﬁrst case, with the support of RES and ESS, we focused on total energy cost optimization. Utilization of energy of the main grid at low price time slot and selling energy were evaluated in terms of total energy cost during a day. In the second case, we minimized our HEMS based on two objectives: total energy cost and PAR. In both cases, our program is run on Intel(R) Core(TM) i7-8700 CPU @ 3.20GHz (12 CPUs) and 16GB RAM with Windows 10 pro. The computational time of our program was about 10 minutes. In our objective function, we have two kinds of variables: continuous variables such as Echarge MG (t), Eload ESS (t) and binary variables that only have 0 or 1 such as O(ai, t). The original PSO algorithm is only correctly operated in the continuous search space. For binary variables, we must use the binary particle swarm optimization (BPSO) given by Kennedy and Eberhart in [25]. In BPSO algorithm, the formula of each particle’s velocity at iteration t + 1 is the same as (37). To update the value of particle xi(t + 1) at iteration t + 1, instead of using velocity as (38), we use a sigmoid function S(.) given by (39). S(vi(t + 1)) = 1 1 + e−vi(t+1) (39) (39) and and and To compare results of our simulation with one of [3], the input parameters of our simulation were employed from [3] including home appliances, day-ahead pricing xi(t + 1) = ( 1 S(vi(t + 1)) > rand(). 0 S(vi(t + 1)) ≤rand(). (40) (40) 49443 49443 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling TABLE 4. Description of the appliances. FIGURE 4. Hourly prices according to DAP signal. TABLE 5. The input parameters of our ESS in the simulation. A. CASE 1: SINGLE-OBJECTIVE OPTIMIZATION In this case, we focus on minimizing total energy cost of our system during a day. We ﬁrstly assume that Psell(t) = PMG(t)∀t which means α = 1. 49444 VOLUME 8, 2020 VOLUME 8, 2020 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling FIGURE 7. Total energy cost of different algorithms [3]. FIGURE 8. Hourly energy of the main grid stored in ESS. 1) TOTAL ENERGY COST The comparison of total energy cost of our system and [3] is shown in Fig. 7. FIGURE 9. Hourly ESS energy used for home load. FIGURE 10. Hourly ESS energy used for selling. TABLE 6. Schedule of the shiftable appliances. FIGURE 9. Hourly ESS energy used for home load. FIGURE 7. Total energy cost of different algorithms [3]. FIGURE 9. Hourly ESS energy used for home load. FIGURE 7. Total energy cost of different algorithms [3]. FIGURE 8. Hourly energy of the main grid stored in ESS. FIGURE 8 Hourly energy of the main grid stored in ESS FIGURE 10. Hourly ESS energy used for selling. FIGURE 8. Hourly energy of the main grid stored in ESS. FIGURE 10. Hourly ESS energy used for selling. TABLE 6. Schedule of the shiftable appliances. TABLE 6. Schedule of the shiftable appliances. 1) TOTAL ENERGY COST In detail, at low price time such as 7 P.M., 8 P.M., and 9 P.M., our system uses a lot of energy from the main grid for home devices. Moreover, at time from 0 A.M. to 7 A.M., because of utilization of cheap energy from the main grid, the load demand from the main grid is bigger than the energy which is required by the home appliances. Whereas, the need of energy of the main grid is very small at high price time such as 7 A.M., 8 A.M. because of the support of RES and ESS. Even our HEMS does not need energy of the main grid at time from 11 A.M. to 1 P.M since our ESS and RES have sufﬁcient energy for home load at these time slots. In detail, at low price time such as 7 P.M., 8 P.M., and 9 P.M., our system uses a lot of energy from the main grid for home devices. Moreover, at time from 0 A.M. to 7 A.M., because of utilization of cheap energy from the main grid, the load demand from the main grid is bigger than the energy which is required by the home appliances. Whereas, the need of energy of the main grid is very small at high price time such as 7 A.M., 8 A.M. because of the support of RES and ESS. Even our HEMS does not need energy of the main grid at time from 11 A.M. to 1 P.M since our ESS and RES have sufﬁcient energy for home load at these time slots. FIGURE 14. Average total energy cost with different Chrate/Dhrate and capacity of ESS. and Table 7 show the average total energy cost of our sys- tem with different Chrate/Dhrate and ESS capacity when we run our simulation with same PSO parameters as shown in Table 3. As shown in Table 7, the total energy cost has a steady decrease with increasing Chrate/Dhrate. In particular, with 3 kWh of ESS capacity, average total energy cost is 431.61 cents and 425 cents for 0.6 kW and 0.9 kW of Chrate/Dhrate respectively. Compared with total energy cost of Chrate/Dhrate = 0.3 kW, the average energy cost of our system is reduced slightly by 3.2% and by 4.7%. 1) TOTAL ENERGY COST Assuming that ESS has an inﬁnite space to store energy (no limit), we get maximum beneﬁt from increasing Chrate/Dhrate parameter with a remarkable drop to 4.3% and 8.5% for 0.6 kW and 0.9 kW of Chrate/Dhrate respectively. In addition, from results of our simulation, ESS capacity required to store enough energy is around 4 kWh and 6.5 kWh respectively. We get these results because with bigger Chrate/Dhrate we can In summary, above three main factors make total energy cost of our system drop signiﬁcantly. However, because we only focus on minimizing the energy cost, the average PAR of our system in this case is higher than algorithms of [3] as illustrated in the Fig. 13. There is a trade-off between decreasing the total energy cost and reducing the system’s PAR. We thus try to balance these values in multi-objective optimization section. 1) TOTAL ENERGY COST The comparison of total energy cost of our system and [3] is shown in Fig. 7. The comparison of total energy cost of our system and [3] is shown in Fig. 7. Total energy cost of our system is 445.78 cents, the small- est energy cost among six algorithms. Compared with [3]’s BPSO algorithm, the total energy cost of our system is signif- icantly reduced by 19.7%. To understand where this beneﬁt comes from, we analyze an output of our simulation for all appliances to see how our HEMS utilizes energy of the main grid and ESS energy as shown in Fig. 8, Fig. 9, and Fig. 10 respectively. reasons: The ﬁrst reason is that RES generates energy at high price times. Hence, immediately using it for home devices is better than storing it in ESS. Furthermore, storing RES energy to ESS and discharging later lead to lose of energy due to round-trip efﬁciency. The second reason is that the amount of energy generated by RES is smaller than energy needed by home devices in all time slots. In this case, our HEMS prefers using it for home devices to storing it in ESS. Firstly, in order to decrease the total energy cost, our HEMS tries to utilize energy of the main grid by storing main grid’s electricity in ESS at low price times such as 0 A.M., 1 A.M., 2 A.M. as shown in Fig. 8. This cheap energy will be used for home devices at high price time such as from 8 A.M. to 9 A.M. and from 9 A.M. to 10 A.M. (Fig. 9) or to sell to the outside at high price time such as from 7 A.M. to 8 A.M. and from 10 A.M. to 11 A.M. (Fig. 10). Finally, Table 6 shows the schedule of each shiftable appli- ance which is the useful result of our algorithm. Our HEMS schedules our appliances to operate at low price time. With this schedule, we have hourly energy needed by home appli- ances (red line) and hourly load demand of our system from the main grid (blue line) at each time as shown in Fig. 12. Secondly, as illustrated by Fig. 11, most of the energy generated from RES is used for home devices for two 49445 VOLUME 8, 2020 H. T. 1) TOTAL ENERGY COST Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling FIGURE 11. RES energy used for ESS storing and home load. FIGURE 12. Hourly load demand from main grid. In detail, at low price time such as 7 P.M., 8 P.M., and 9 P.M., our system uses a lot of energy from the main grid for home devices. Moreover, at time from 0 A.M. to 7 A.M., because of utilization of cheap energy from the main grid, the load demand from the main grid is bigger than the energy which is required by the home appliances. Whereas, the need of energy of the main grid is very small at high price time such as 7 A.M., 8 A.M. because of the support of RES and ESS. Even our HEMS does not need energy of the main grid at time from 11 A.M. to 1 P.M since our ESS and RES have sufﬁcient energy for home load at these time slots. In summary, above three main factors make total energy cost of our system drop signiﬁcantly. However, because we FIGURE 13. PAR of different algorithms [3]. FIGURE 14. Average total energy cost with different Chrate/Dhrate and capacity of ESS. and Table 7 show the average total energy cost of our sys- tem with different Chrate/Dhrate and ESS capacity when we run our simulation with same PSO parameters as shown in Table 3. As shown in Table 7, the total energy cost has a steady decrease with increasing Chrate/Dhrate. In particular, with 3 kWh of ESS capacity, average total energy cost is 431.61 cents and 425 cents for 0.6 kW and 0.9 kW of , e e d FIGURE 13. PAR of different algorithms [3]. FIGURE 14. Average total energy cost with different Chrate/Dhrate and capacity of ESS. FIGURE 13. PAR of different algorithms [3]. FIGURE 11. RES energy used for ESS storing and home load. FIGURE 11. RES energy used for ESS storing and home load. FIGURE 11. RES energy used for ESS storing and home load. FIGURE 12. Hourly load demand from main grid. FIGURE 13. PAR of different algorithms [3]. FIGURE 1 A t t l t ith diff t Ch /Dh d FIGURE 12. Hourly load demand from main grid. 3) OUR SYSTEM WITH SMALLER SELLING PRICE In this subsection, the effect of smaller selling prices on total energy cost is considered. If energy generated from RES is always smaller than load demand of home appliances and selling price is smaller than price of the main grid at all time during a day, no energy should be sold to the outside. Hence, for this subsection, the area of solar cells is doubled to double the amount of RES energy at all time slots. To store all energy from RES, our ESS has Chrate/Dhrate = 0.9 kW and its capacity is always enough to store energy from RES and the main grid (no limit). Table 8 shows the average total energy cost of our system with α = 1, α = 0.9 and α = 0.8. It means that we consider three cases with Psell(t) = PMG(t), Psell(t) = 0.9 × PMG(t), and Psell(t) = 0.8 × PMG(t)∀t respectively. FIGURE 16. Hourly selling energy with double area of solar cells. time from 7 A.M. to 11 A.M. (Fig. 16) or for home load at time from 7 A.M. to 8 A.M. (Fig. 17). It is worth noting that energy stored in ESS comes from two sources: energy from RES and energy of main grid at low price time (Fig. 18). With the support of cheap energy, the selling energy is maximum with 0.9 kWh at time from 8 A.M. to 11 A.M. With double amount of energy from RES, our average energy cost decreases by 26% from 407.69 cents (Table 7) to 301.66 cents (Table 8). In addition, the average energy cost of our system is increased when selling price is decreased. In particular, if selling price Psell(t) is reduced by 10%, the average energy cost rises to around 2.6%. At time from 11 A.M. to 1 P.M., ESS is set to the charge mode after selling. With this mode, the surplus energy of RES, after providing for home load, is stored in ESS. At these time slots, the selling energy and ESS energy for home load are zero because ESS energy can not be discharged. At time from 1 P.M. to 3 P.M., ESS is set to the discharge mode. Hence, ESS energy can be drawn to sell to the outside and provide for home load again as shown in Fig. 16 and Fig. 17, respectively. 2) OUR SYSTEM WITH DIFFERENT ESS In this subsection, we consider the effects of Chrate/Dhrate and capacity of ESS on minimizing the energy cost. Fig. 14 49446 VOLUME 8, 2020 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling FIGURE 15. Hourly RES energy with double area of solar cells. FIGURE 16. Hourly selling energy with double area of solar cells. time from 7 A.M. to 11 A.M. (Fig. 16) or for home load at time from 7 A.M. to 8 A.M. (Fig. 17). It is worth noting that energy stored in ESS comes from two sources: energy from RES and energy of main grid at low price time (Fig. 18). With TABLE 7. Average total energy cost with different Chrate/Dhrate and capacity of ESS. TABLE 8. Average total energy cost with different selling prices. TABLE 7. Average total energy cost with different Chrate/Dhrate and capacity of ESS. TABLE 8 A t t l t ith diff t lli i TABLE 7. Average total energy cost with different Chrate/Dhrate and capacity of ESS. TABLE 8. Average total energy cost with different selling prices. TABLE 8. Average total energy cost with different selling prices. ABLE 8. Average total energy cost with different selling price store more energy of the main grid at low price time slot and use it for higher price time slot. Furthermore, Table 7 shows a trend to decrease PAR of our system when the Chrate/Dhrate is increased. FIGURE 15. Hourly RES energy with double area of solar cells. FIGURE 15. Hourly RES energy with double area of solar cells. FIGURE 16. Hourly selling energy with double area of solar cells. FIGURE 16. Hourly selling energy with double area of solar cells. The increasing of Chrate/Dhrate helps to considerably decrease the average total energy cost of our system. How- ever, to get maximum beneﬁt from this increase, we must have an ESS with sufﬁcient capacity to store energy. 3) OUR SYSTEM WITH SMALLER SELLING PRICE To see how our HEMS utilizes the RES energy and energy of the main grid in this case, we analyze an output of the best case of our simulation with α = 0.9 as shown in Fig. 15, Fig. 16, Fig.17 and Fig. 18. As depicted in Fig. 15, at peak-price time from 7 A.M. to 11 A.M., most of energy generated by RES is used for home load. Because RES energy is larger than energy demand from home devices at time from 8 A.M. to 11 A.M., the sur- plus energy is lost. The lost energy can not be stored in ESS because ESS is set to discharge mode. With this mode, energy from ESS can only be drawn out to sell to the outside at B. CASE 2: MULTI-OBJECTIVE OPTIMIZATION PAR describes the behavior of the consumer’s home load and it affects the operation of the main grid. As described 49447 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling FIGURE 17. Hourly ESS energy for home load with double area of solar cells. FIGURE 18. Hourly energy of the main grid stored in ESS with double area of solar cells. TA FI ot si PA av (w de to of ha av ca FIGURE 17. Hourly ESS energy for home load with double area of solar cells. TABLE 9. Average PAR and total energy cost with different w2. FIGURE 19. Average PAR and average energy cost with different w2 and other algorithms. TABLE 9. Average PAR and total energy cost with different w2. FIGURE 17. Hourly ESS energy for home load with double area of solar cells. FIGURE 18. Hourly energy of the main grid stored in ESS with double area of solar cells. FIGURE 18. Hourly energy of the main grid stored in ESS with double area of solar cells FIGURE 19. Average PAR and average energy cost with different w2 and other algorithms. simulation, we keep w1 = 1 and change w2 to achieve the PAR we desire. Fig. 19 and Table 9 show the average total energy cost and average PAR of our system in 3 cases: (w1 = 1, w2 = 10), (w1 = 1, w2 = 20), and (w1 = 1, w2 = 30). A steady decrease in average PAR and a gradual increase in average total energy cost were observed with increasing w2. More speciﬁcally, with (w1 = 1, w2 = 10), average PAR of our system is 2.396, a decrease of 17.4% whereas we have a 6% increase in average energy cost, as compared with average PAR and total energy cost of our system in the SOO case. In this case, we still have higher average PAR than [3]’s BPSO algorithm. With (w1 = 1, w2 = 20), average PAR of our system is 2.076, a decrease of 28.4% compared with PAR of our system in the SOO case whereas our system’s average total energy cost is only 496.84 cents. Compared to PAR and total energy cost of [3]’s BPSO algorithm, these values of our system is remarkable. B. CASE 2: MULTI-OBJECTIVE OPTIMIZATION Simulation r values of weight const our system can be de energy cost and PAR of with w1 = 1, w2 = 2 system were reduced ESS parameters, simul considerably reduction of ESS are increased. E by 4.3% and 8.5% with respectively and ESS m energy. With development of ment architecture in glo main responsibilities a energy reliability unde demand of prosumers. I with this system to imp In our future system, comforts, such as therm also be considered. Rea improve our system. W optimized with real-tim REFERENCES [1] G. Dileep, ‘‘A survey on Energy, vol. 146, pp. 258 TABLE 10. Schedule of the Shiftable appliances in MOO best case with w2 = 20. FIGURE 20. Hourly load demand of MOO best case from main grid with w2 = 20. TABLE 10. Schedule of the Shiftable appliances in MOO best case with w2 = 20. and PAR. Simulation results showed that, with appropriate values of weight constants w1, w2, energy cost and PAR of our system can be decreased to values smaller than both energy cost and PAR of BPSO algorithm in [3]. In particular, with w1 = 1, w2 = 20, both energy cost and PAR of our system were reduced by approximately 10%. In terms of ESS parameters, simulation results also show that there is considerably reduction when Chrate/Dhrate and the capacity of ESS are increased. Energy cost of our system was reduced by 4.3% and 8.5% with 0.6 kW and 0.9 kW of Chrate/Dhrate respectively and ESS must have sufﬁcient capacity to store energy. With development of HEMSs, user-mode energy manage- ment architecture in global scale is suggested for SG [26]. Its main responsibilities are to maintain energy efﬁciency and energy reliability under uncertain electricity generation and demand of prosumers. In future, our HEMS need to cooperate with this system to improve operations of SG. In our future system, besides electricity cost and PAR, user comforts, such as thermal comfort and consecutive tasks, will also be considered. Real-time optimization is another way to improve our system. With this technology, our system can be optimized with real-time usage data. REFERENCES FIGURE 20. Hourly load demand of MOO best case from main grid with w2 = 20. [1] G. Dileep, ‘‘A survey on smart grid technologies and applications,’’ Renew. Energy, vol. 146, pp. 2589–2625, Feb. 2020. [2] B. P. Esther and K. S. Kumar, ‘‘A survey on residential demand side management architecture, approaches, optimization models and methods,’’ Renew. Sustain. Energy Rev., vol. 59, pp. 342–351, Jun. 2016. As depicted in this ﬁgure, the biggest energy needed from the main grid is approximately 4.2 kWh from 7 P.M. to 8 P.M. and this value is spread through many time slots. This is a signiﬁcant decrease as comparing with the biggest load demand of SOO case of 6 kWh in the Fig. 12. This result is the main reason help our system’s PAR to decrease. At peak hours such as from 4 P.M. to 6 P.M., with the support of RES and ESS, the load demand from main grid also goes down, compared with energy needed for home appliances. This support also makes our PAR decrease. [3] A. Ahmad, A. Khan, N. Javaid, H. M. Hussain, W. Abdul, A. Almogren, A. Alamri, and I. Azim Niaz, ‘‘An optimized home energy management system with integrated renewable energy and storage resources,’’ Energies, vol. 10, no. 4, p. 549, 2017. [4] J. S. Vardakas, N. Zorba, and C. V. Verikoukis, ‘‘A survey on demand response programs in smart grids: Pricing methods and optimization algorithms,’’ IEEE Commun. Surveys Tuts., vol. 17, no. 1, pp. 152–178, 1st Quart., 2015. [5] P. Siano, ‘‘Demand response and smart grids—A survey,’’ Renew. Sustain. Energy Rev., vol. 30, pp. 461–478, Feb. 2014. [6] J. Y. Lee and S. G. Choi, ‘‘Linear programming based hourly peak load shaving method at home area,’’ in Proc. 16th Int. Conf. Adv. Commun. Technol., Feb. 2014, pp. 310–313. VII. CONCLUSIONS AND FUTURE WORKS B. CASE 2: MULTI-OBJECTIVE OPTIMIZATION By using MOO, our system has better performance in both elements: PAR and total energy cost. Our average PAR is smaller than the PAR of [3]’s BPSO algorithm by approximately 10.2% and our average energy cost is smaller than the energy cost of [3]’s BPSO algorithm by approximately 10.5%. FIGURE 18. Hourly energy of the main grid stored in ESS with double area of solar cells. in previous section, when we only focus on minimizing the total energy cost, our system’s PAR remains very high as shown in Fig. 13. To solve this problem, we try to mini- mize two aspects: total energy cost and PAR at the same time. By using weight method of multi-objective optimization (MOO), we have a new objective function. min(w1 × Cday + w2 × PAR) (41) (41) where Cday is calculated by (34) and w1 is the weight of variable Cday and is a constant. PAR is calculated by (36) and w2 is the weight of variable PAR and is also a constant. Table 10 shows the schedule of shiftable appliances in MOO best case of our system with w2 = 20. The pro- posed HEMS schedules the appliances at appropriate time to decrease the PAR. It is worth noting that there is a big difference with the schedule of our appliances in SOO case (Table 6). From this schedule, we have hourly energy which is needed by home appliances and hourly load demand of our system from the main grid as shown in the Fig. 20. The input parameters of our simulation are the same as the single-objective optimization (SOO). Because of the compli- cated objective function, the running time of our simulation is slightly increased to 11.5 minutes. In this section, w2 is set to a value bigger than the value of w1 with the hope that PAR is decreased while Cday is increased to acceptable value. In our 49448 49448 VOLUME 8, 2020 VOLUME 8, 2020 H. T. Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling TABLE 10. Schedule of the Shiftable appliances in MOO best case with w2 = 20. FIGURE 20. Hourly load demand of MOO best case from main grid with w2 = 20. and PAR. [1] G. Dileep, ‘‘A survey on smart grid technologies and applications,’’ Renew. Energy, vol. 146, pp. 2589–2625, Feb. 2020. VII. CONCLUSIONS AND FUTURE WORKS degrees in electrical engineering from Yonsei Uni- versity, Seoul, South Korea, in 2007 and 2014, respectively. From 2014 to 2018, he was with Aalborg University, Aalborg, Denmark, as a Post- doctoral Researcher. Since 2018, he has been with the Department of the Internet of Things, Soonchunhyang University, Asan, South Korea, where he is currently an Assistant Professor. His research interests include machine-to-machine communications, ultra-reliable low-latency communications, cross-layer optimization in wireless networks, network information theory, networked robotics, economic aspect of wireless network technologies, the Internet of Things, and distributed machine learning. He has served as a Technical Program Committee Member for the IEEE Globecom 2015 Workshop on Optimizing Heterogeneous Networking Technologies for the Internet of Things. [19] C. Keles and Y. Yigit, ‘‘Implementation of demand side load management by domestic load shedding algorithm for conventional electricity meters,’’ in Proc. IDAP, Sep. 2019, pp. 1–4. [20] J. Yoo, B. Park, K. An, E. A. Al-Ammar, Y. Khan, K. Hur, and J. H. Kim, ‘‘Look-ahead energy management of a grid-connected residential PV sys- tem with energy storage under time-based rate programs,’’ Energies, vol. 5, no. 4, pp. 1116–1134, 2012. [21] D. Fuselli, F. De Angelis, M. Boaro, S. Squartini, Q. Wei, D. Liu, and F. Piazza, ‘‘Action dependent heuristic dynamic programming for home energy resource scheduling,’’ Int. J. Electr. Power Energy Syst., vol. 48, pp. 148–160, Jun. 2013. [22] Y. Ru, J. Kleissl, and S. Martinez, ‘‘Storage size determination for grid- connected photovoltaic systems,’’ IEEE Trans. Sustain. Energy, vol. 4, no. 1, pp. 68–81, Jan. 2013. [23] M. Amer, A. Naaman, N. K. M’Sirdi, and A. M. El-Zonkoly, ‘‘Smart home energy management systems survey,’’ in Proc. Int. Conf. Renew. Energies Developing Countries, Nov. 2014, pp. 167–173. [24] R. Eberhart and J. Kennedy, ‘‘Particle swarm optimization,’’ in Proc. IEEE Int. Conf. Neural Netw., vol. 4, 1995, pp. 1942–1948. [25] J. Kennedy and R. C. Eberhart, ‘‘A discrete binary version of the particle swarm algorithm,’’ in Proc. IEEE Int. Conf. Syst., Man, Cybern. Comput. Cybern. Simulation, vol. 5, Oct. 1997, pp. 4104–4108. KYU-HAENG LEE received the B.S. degree in computer science and technology from Tsinghua University, Beijing, China, and the Ph.D. degree in computer science and engineering from Seoul National University, in 2009 and 2015, respec- tively. He was a Senior Software Engineer with Samsung Research, Seoul, South Korea, from 2015 to 2018. VII. CONCLUSIONS AND FUTURE WORKS Dinh et al.: HEMS With Renewable Energy and Energy Storage Utilizing Main Grid and Electricity Selling JAESEOK YUN received the B.S. degree in elec- tronics engineering from Chonnam National Uni- versity, in 1997, and the M.S. and Ph.D. degrees in mechatronics from the Gwangju Institute of Sci- ence and Technology (GIST), in 1999 and 2006, respectively. He worked as a Senior Researcher with the IoT Platform Research Center, Korea Electronics Technology Institute (KETI), from 2009 to 2016. He also worked as a Postdoctoral Research Scientist with the Ubiquitous Computing Research Group, School of Interactive Computing, Georgia Institute of Technology, USA, from 2006 to 2009. He is currently an Assistant Professor with the Department of the Internet of Things, Soonchunhyang University. His research interests include ubiquitous computing, the Internet of Things (IoT), and machine learning-enabled applications in everyday lives. [13] H. Hussain, N. Javaid, S. Iqbal, Q. Hasan, K. Aurangzeb, and M. Alhussein, ‘‘An efﬁcient demand side management system with a new optimized home energy management controller in smart grid,’’ Energies, vol. 11, no. 1, p. 190, 2018. [14] O. Samuel, S. Javaid, N. Javaid, S. Ahmed, M. Afzal, and F. Ishmanov, ‘‘An efﬁcient power scheduling in smart homes using jaya based opti- mization with Time-of-Use and critical peak pricing schemes,’’ Energies, vol. 11, no. 11, p. 3155, 2018. [15] S. Aslam, Z. Iqbal, N. Javaid, Z. Khan, K. Aurangzeb, and S. Haider, ‘‘Towards efﬁcient energy management of smart buildings exploiting heuristic optimization with real time and critical peak pricing schemes,’’ Energies, vol. 10, no. 12, p. 2065, 2017. Energies, vol. 10, no. 12, p. 2065, 2017. [16] A. Anvari-Moghaddam, A. Rahimi-Kian, M. S. Mirian, and J. M. Guerrero, ‘‘A multi-agent based energy management solution for integrated buildings and microgrid system,’’ Appl. Energy, vol. 203, pp. 41–56, Oct. 2017. [17] M. Awais, N. Javaid, K. Aurangzeb, S. Haider, Z. Khan, and D. Mahmood, ‘‘Towards effective and efﬁcient energy management of single home and a smart community exploiting heuristic optimization algorithms with critical peak and real-time pricing tariffs in smart grids,’’ Energies, vol. 11, no. 11, p. 3125, 2018. [18] C. Keles, A. Karabiber, M. Akcin, A. Kaygusuz, B. B. Alagoz, and O. Gul, ‘‘A smart building power management concept: Smart socket applica- tions with DC distribution,’’ Int. J. Electr. Power Energy Syst., vol. 64, pp. 679–688, Jan. 2015. DONG MIN KIM received the B.S. and Ph.D. VII. CONCLUSIONS AND FUTURE WORKS [7] K. C. Sou, J. Weimer, H. Sandberg, and K. H. Johansson, ‘‘Schedul- ing smart home appliances using mixed integer linear programming,’’ in Proc. IEEE Conf. Decis. Control Eur. Control Conf., Dec. 2011, pp. 5144–5149. This study presented a new HEMS with integration of RES and ESS. Our objective was to minimize energy cost and PAR of our system during a day. The algorithms we used in our system were the combination of PSO and BPSO. Our HEMS is able to utilize electricity of the main grid at low price time to provide for home appliances at high price time with the support of ESS and RES. In addition, our HEMS support selling electricity to the outside. To achieve our objec- tive, we built general mathematical formulas for energy cost and PAR and evaluated our HEMS by performing extensive simulations. With new functions, energy cost of our HEMS was signiﬁcantly reduced to 19.7%, as compared to previous results of BPSO algorithms in [3]. However, when our system only focuses on the minimization of energy cost, PAR of our system remains very high. In order to reduce PAR, we used the weighted method of MOO to minimize both energy cost [8] F. Y. Melhem, O. Grunder, Z. Hammoudan, and N. Moubayed, ‘‘Opti- mization and energy management in smart home considering photovoltaic, wind, and battery storage system with integration of electric vehicles,’’ Can. J. Elect. Comput. Eng., vol. 40, no. 2, pp. 128–138, Aug. 2017. [9] A. Anvari-Moghaddam, H. Monsef, and A. Rahimi-Kian, ‘‘Optimal smart home energy management considering energy saving and a comfort- able lifestyle,’’ IEEE Trans. Smart Grid, vol. 6, no. 1, pp. 324–332, Jan. 2015. [10] A. Anvari-Moghaddam, J. M. Guerrero, J. C. Vasquez, H. Monsef, and A. Rahimi-Kian, ‘‘Efﬁcient energy management for a grid-tied resi- dential microgrid,’’ IET Gener., Transmiss. Distrib., vol. 11, no. 11, pp. 2752–2761, Aug. 2017. [11] P. Samadi, V. W. S. Wong, and R. Schober, ‘‘Load scheduling and power trading in systems with high penetration of renewable energy resources,’’ IEEE Trans. Smart Grid, vol. 7, no. 4, pp. 1802–1812, Jul. 2016. [12] K. Ma, T. Yao, J. Yang, and X. Guan, ‘‘Residential power scheduling for demand response in smart grid,’’ Int. J. Electr. Power Energy Syst., vol. 78, pp. 320–325, Jun. 2016. 49449 49449 VOLUME 8, 2020 H. T. VII. CONCLUSIONS AND FUTURE WORKS He is currently an Assistant Pro- fessor with the Department of the IoT, Soonchun- hyang University. His research interests include MIMO/OFDM systems, the IEEE 802.11, system optimization, and the IoT. [26] B. B. Alagoz, A. Kaygusuz, and A. Karabiber, ‘‘A user-mode distributed energy management architecture for smart grid applications,’’ Energy, vol. 44, no. 1, pp. 167–177, Aug. 2012. HUY TRUONG DINH (Student Member, IEEE) was born in Quang Nam, Viet Nam, in 1981. He received the B.S. degree in information tech- nology from the Posts and Telecommunications Institute of Technology, Ho Chi Minh, Viet Nam, in 2004, and the M.S. degree in computer engineer- ing from the Delft University of Technology, Delft, The Netherlands, in 2009. He is currently pursuing the Ph.D. degree with Soonchunhyang University, Asan, South Korea. His research interest includes the IoT architecture, the IoT in energy, and blockchain. His awards and hon- ors include Top Talent Fellowship of EEMCS faculty of the Delft University of Technology, the First Prize of Student Science Research of the Posts and Telecommunications Institute of Technology. DAEHEE KIM (Member, IEEE) received the B.S. degree in electrical and electronic engineer- ing from Yonsei University, Seoul, South Korea, in 2003, and the M.S. and Ph.D. degrees in electri- cal and electronic engineering from Korea Univer- sity, Seoul, in 2006 and 2016, respectively. From 2006 to 2016, he was a Senior Engineer with Sam- sung Electronics, Suwon, South Korea, where he conducted research on WiMAX and LTE systems. He is currently an Assistant Professor with the Department of the Internet of Things, Soonchunhyang University, Asan, South Korea. His research interest includes the Internet of Things, energy management, blockchain, 5G, and security for wireless networks. VOLUME 8, 2020 49450 VOLUME 8, 2020
W2153493434.txt	https://www.animbiosci.org/upload/pdf/ajas-27-11-1663.pdf	en		Farmers’ Perceptions and Knowledge of Cattle Adaptation to Heat Stress and Tick Resistance in the Eastern Cape, South Africa	Asian-Australasian journal of animal sciences	2,014	cc-by	5,244	1663 Open Access Asian Australas. J. Anim. Sci. Vol. 27, No. 11 : 1663-1670 November 2014 http://dx.doi.org/10.5713/ajas.2014.14174 www.ajas.info pISSN 1011-2367 eISSN 1976-5517 Farmers’ Perceptions and Knowledge of Cattle Adaptation to Heat Stress and Tick Resistance in the Eastern Cape, South Africa C. L. F. Katiyatiya, V. Muchenje, and A. Mushunje1 Department of Livestock and Pasture Science, University of Fort Hare, Alice 5700, South Africa ABSTRACT: The objective of this study was to determine the perceptions and knowledge of farmers of heat stress and tick resistance in cattle. A cross-sectional survey was conducted and 110 farmers in four villages in the sour and sweet velds of the Eastern Cape Province, South Africa were interviewed. The associations among area (municipality), gender, age, level of education, employment and religion were computed using Chi-square tests. The majority of the respondents had on average 4 bulls, 4 cows, 4 heifers, 4 calves, and 4 oxen. Milk was considered as the major (28.3%) reason for keeping cattle. Most farmers owned non-descript (72.6%), and Nguni (45.3%) cattle because of their heat tolerance (54.7%), tick resistance (54.7%), and milking ability (28.2%) traits. Excessive panting (56.6%) and disease transmission (76%) were regarded as the major effects of heat stress and tick infestation in cattle, respectively. About 50% of the respondents agreed that hair length influences tick resistance and 47.17% considered coat colour when acquiring cattle. In the sampled areas, ticks were prevalent in the summer season (93%), and 77.36% of the respondents use acaricides every fortnight. Gall sickness was reported to be a major problem in the cattle herds by 36.79% of the respondents. Our results showed that farmers in the two municipalities had knowledge of cattle adaptation to heat stress and tick resistance. (Key Words: Herd Composition, Nguni, Non-descript Genotype, Season, Socio-demography) INTRODUCTION Sustainable livestock production is very important to the livelihoods of people and therefore there is need to improve it (Lamy et al., 2012). According to Thornton (2010), about 600 million farmers in the communal areas have their livelihoods largely supported by livestock production. It is of great importance for farmers to keep animals that are well adapted to their environmental conditions in order to maximize production. Farmers are keen on keeping animals that provide them with good returns. Cattle, goats, sheep and chickens kept by farmers in the communal areas allow them to have a source of income, draught power, manure and other socio-economic activities to cater for their day to day needs (Mapiye et al., 2009a; Nqeno et al., 2011). These functions and roles differ with the socio-economic factors, Corresponding Author: V. Muchenje. Tel: +27-40-602-2059, Fax: +27-84-422-1487, E-mail: vmuchenje@ufh.ac.za 1 Department of Agricultural Economics and Extension, University of Fort Hare, Alice 5700, South Africa. Submitted Mar. 6, 2014; Revised Apr. 30, 2014; Accepted Jun. 2, 2014 cultural and biological attributes of a community. Communal areas are largely dominated by small scale and resource-limited farmers. These areas have vast arable and grazing lands suitable for indigenous breeds. The Nguni cattle breed is ideal because it is indigenous and is known for its ability to adapt well in low grazing communal areas (Muchenje et al., 2008a; Musemwa et al., 2010). The breed’s remarkable attributes have seen its popularity increasing in the South African rural communities. Musemwa et al. (2010) described the Nguni breed as being environmentally friendly since it requires low management and maintenance inputs. As a result, the Nguni cattle development project in the Eastern Cape Province, South Africa has contributed towards the introduction of this breed in many communities thus contributing to poverty alleviation from the benefits of keeping the breed (Mapiye et al., 2007). Variations in climatic and environmental conditions have a major influence on cattle performance and play a big role in determining productivity. Climatic conditions differ from one area to the other and rainfall and temperature variations determine the Copyright © 2014 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/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 1664 Katiyatiya et al. (2014) Asian Australas. J. Anim. Sci. 27:1663-1670 availability of suitable grazing areas for the cattle (Mapiye et al., 2010). High temperatures and humidity favour the breeding of parasites with high tick loads seen after heavy rains (Muchenje et al., 2008b; Lorusso et al., 2013). Such temperature conditions contribute to heat stress incidence whose effects may be deleterious if precautious measures are not undertaken. There is therefore need for farmers to equip themselves with the necessary knowledge, skills and management tactics to boost the performance of their animals in any environment. Farmers are the key players in agricultural production and their knowledge on sustainable agriculture is essential (Chunyan et al., 2013). The perceptions and knowledge they have on animal production are likely to influence how best they will breed their animals. These will also aid in the development of strategic methods and policies that can be implemented to control the devastating effects of problems such as heat stress, ticks and tick-borne diseases. This will in turn enhance production potentials of their animals in the livestock industry. Therefore, the objective of this study was to determine the perceptions and knowledge of farmers’ livelihoods and cattle adaptation to heat stress and tick infestation. and information required that aided in the quality of the survey and identifying the necessary farmers for the study. A structured questionnaire was used to collect information from the farmers about their perceptions and knowledge on adaptation of cattle to heat and tick resistance. Respondents were selected from the communities based on the number of cattle owned and preference was made for those with 4 or more cattle per household. Trained enumerators aided in interviewing the farmers using isiXhosa, the vernacular language of the area. To eliminate bias, open-ended and closed questions were used to acquire information on household, farmers’ livelihood and socio-economic status and respective cattle production. Statistical analysis The PROC FREQ procedures of Statistical Analyses Systems (SAS) (2003) were used to determine the frequencies of gender, age, level of education, employment and religion of the farmers. The Chi-square tests of SAS (2003) were used to determine associations between demographic information and reasons for keeping cattle and perceptions on ticks and heat stress. RESULTS MATERIALS AND METHODS Description of study sites The study was conducted in four communities (Khayamnandi, Nselamanzi, Thanga, and Zazulwana) in the Eastern Cape Province, the second largest in South Africa. The provinces experiences varied climatic conditions as it has inland and coastal areas. Khayamnandi and Nselamanzi are fall under Nkonkobe municipality while Thanga and Zazulwana form part of the Mnquma municipality. Khayamnandi and Nselamanzi are inland areas characterised by a semi-intensive cattle production system in the sweetveld. They are situated at an altitude of 450 to 500 m with mean annual rainfall of 480 mm and 18.7°C mean annual temperature. Thanga and Zazulwana villages are coastal areas mainly characterised by extensive (communal) cattle production on sourveld land. It receives mean rainfall of 596 mm, mainly in summer with mean summer temperature of 25.6°C and mean winter temperature of 19.2°C (South African Explorer, 2014). The villages are located at 700 to 900 m above sea level and under the Grassland biome. Data collection A total of 36, 19, 19, and 36 respondents in Zazulwana, Thanga, Khayamnandi and Nselamanzi communities, respectively, participated in the survey. Informal interviews were done with the extension officers to obtain more data Farmers’ social demographic information The municipality with the highest number of farmer participants was Nkonkobe (54%) while Mnquma municipality had 46%. Table 1 shows the socio-economic dynamics of the 110 farmers who participated in the study. The majority of the farmers (64%) were males. The highest numbers of farmers were above 51 years of age (67%). Most of the farmers (43%) had gone through secondary education (Grade 8 to 12) as their highest level of education and depended on grants/pensions (34%) for income. Most of the farmers (75%) were Christians. Herd composition and uses of cattle The proportion of farmers, herd size and cattle numbers per composition are represented in Figure 1. The proportion of farmers who had ≤4 bulls, cows, heifers, oxen and calves in their herds was 99%, 68%, 86%, 92%, 83%, respectively. Only 4% of the farmers had 15 to 19 cows. Most farmers revealed that, they kept cattle for milk (28%), sales (26%) and ceremonies (11%) as shown in Table 2. The breed(s) and reasons for keeping them are highlighted in Table 3. Some of the farmers kept more than one type of cattle breed. The non-descript cattle (72.6%) were commonly kept by the farmers, followed by the Nguni (45.3%). The majority of the farmers (55%) claimed that tick resistance and heat tolerance attributes were the reasons why they kept certain cattle breeds. 1665 Katiyatiya et al. (2014) Asian Australas. J. Anim. Sci. 27:1663-1670 Table 1. Demography of cattle farmers in the Nkonkobe and Mnquma municipalities in the Eastern Cape Province Proportion (%) 100 64.15 35.85 100 6.60 26.42 30.19 36.79 100 16.04 43.40 35.85 4.72 100 Proportion of farmers (%) Items Gender Male Female Total Age 20-29 30-50 51-60 >60 Total Level of education Tertiary Secondary Primary No formal education Total Employment Farming Employed Self employed Grant/ pension receiver Total Religion Christianity Traditionalism Other Total 120 A - Bulls B - Cows C - Heifers D - Calves E - Oxen/Steers 80 60 40 20 0 ABCDE <4 AB C D E 5-9 BCD 10-14 B 15-19 Cattle numbers Figure 1. Farmers’ herd compositions and sizes (percentages). 27.36 13.21 25.47 33.96 100 74.53 24.53 0.94 100 Farmers’ perceptions and knowledge on coat characteristics, heat stress and tick infestation in cattle The perceptions of farmers on the effects of heat stress on cattle production are shown in Figure 2. A total of 57% of the farmers perceived that heat stress causes excessive panting in cattle, while 48% and 46% reported loss of weight and reduced milk production, respectively. About 70% of the cattle farmers perceived reduced feed intake and death effects of heat stress. The least perceived notion on the effects of heat stress was said to be reduced breeding efficiency stated by 26.42% of the farmers. The perceptions and knowledge of farmers on the effects of ticks is highlighted in Figure 3. Most farmers (76%) reported that ticks cause the transmission of tick-borne diseases. The results show that 33.02% of the farmers consider tick worry, 30.19% consider undesired meat quality and 21.7% reported death to be effects of ticks in cattle. As indicated in Figure 4, a total of 47.17% of the farmers consider coat colour when acquiring their cattle and 38.68% have knowledge on cattle adaptation to heat stress. About 50% of the farmers agreed that hair allow ticks to attach easily to the skin of cattle and 33.96% highlighted dark coat colour relates to more tick loads in cattle. Only 30.19% were positive that cattle with long hairs experience heat stress less often and 21.7% mentioned that darker colours absorb more heat allowing the cattle to be prone to effects of heat stress. Farmers’ perceptions and knowledge on seasonal tick prevalence, tick-borne diseases, and tick control methods and practices The responses of farmers to seasons of tick prevalence are shown in Figure 5. The majority of farmers (93%) reported summer as the season when tick infestation is highest in cattle. Most of the farmers (77%) use acaricides, (12%) make use of ethno-veterinary medicines and (11%) use both acaricides and ethno-veterinary medicines to control ticks in their herds of cattle (Figure 6). The frequency of tick dipping on a yearly basis (Figure 7) was every fortnight in summer and once a month in winter as reported by 62% of the farmers. About 29% of the farmers dip their cattle monthly throughout the year. The tick-borne Table 2. Proportion (%) of various uses of cattle in the Eastern Cape Province, South Africa Socio-economic purpose Frequency Proportion (%) Skin 2 1.89 Manure 4 3.77 Draught 8 7.55 Meat 10 9.43 Milk 30 28.3 Status quo 7 6.6 Dowry (lobola) 5 4.72 Ceremonies 12 11.32 Sales 28 26.42 1666 Katiyatiya et al. (2014) Asian Australas. J. Anim. Sci. 27:1663-1670 Table 3. Cattle breeds kept by farmers and reasons for keeping the specific breeds Breed1 Frequency Brahman 22 Bonsmara 5 Angus 1 Nguni 48 Shorthorn 5 Non-descript 77 Reason for keeping specific breed(s) Heat tolerance 58 Milking ability 33 Tick resistance 58 Meat quality 25 Temperament 23 Fertility 28 Traditionalism (ugqirha) 1 1 Percentage 18.8 4.7 0.9 45.3 4.7 72.6 54.7 28.2 54.7 23.6 21.7 26.4 0.9 Means some of the farmers kept more than one cattle breed. diseases’ prevalence is shown in Figure 8. About 37% and 26% of the farmers reported that their cattle had suffered from gall sickness and red water, respectively. A total of (20.75%) farmers had their cattle suffer from heart water. Of all the farmers (16.04%) never experienced tick-borne disease problems in their animals. Association of farmers’ demography, reasons for keeping cattle and their perceptions and knowledge on heat stress and ticks The association between demographic information of the farmers and reasons for keeping certain cattle breeds are shown in Table 4. Area, gender and religion of the farmers influenced their perceptions on heat tolerance. Tick Figure 2. Farmers’ perceptions and knowledge on heat stress and its implications on cattle. Figure 3. Farmers’ perceptions and knowledge on ticks and their implications on cattle. tolerance perception was associated with area. On the other hand, level of education and employment played a role in the meat quality views of the farmers. The perception on fertility was associated with area, while the level of education influenced the farmers’ knowledge on cattle adaptation to heat stress. The results highlighted in Table 5 show that location of farm significantly (p<0.05) influenced the perceptions on excessive panting, death and coat colour in heat stressed cattle. The opinions of the farmers on coat colour were different (p<0.05) for gender. Hair length perceptions were determined by the farmers’ level of education. The association of the demographic information of the farmers with the knowledge on effects of ticks in cattle is indicated in Table 6. Location of farm was associated with the perception that, ticks cause reduced feed intake, disease transmission and that coat colour plays a role in the extent of infestation. Perceptions and knowledge on ticks was influenced by gender as more males than females Figure 4. Farmers’ perceptions on coat characteristics and their relation to heat stress and ticks. Katiyatiya et al. (2014) Asian Australas. J. Anim. Sci. 27:1663-1670 1667 Figure 5. Farmers’ perceptions and knowledge of seasonal tick prevalence. revealed that, tick infestation causes tick worry and reduced feed intake in cattle. DISCUSSION From this study, men owned more cattle as compared to women. This concurs with Mapiye et al. (2009a) and Tada et al. (2013) who reported that men are usually the owners of large stock such as cattle. Similar findings were observed in Nigeria and Tanzania by Kristjanson et al. (2010) and Covarrubias et al. (2012) where men dominated the livestock industry in rural areas as they were in possession of more cattle than women. This indicates that, there are other responsibilities for women that may not be associated with livestock production such as household chores (Musemwa et al., 2010; Fayemi and Muchenje, 2013). These results further influence the number of cattle in areas that may be having more women than men. Over the years, women have been encouraged to participate actively even in male dominated industries to enable their empowerment and development of communities and these Figure 6. Methods used by farmers to control tick infestation in cattle. Figure 7. Farmers’ response to how often they practice tick control on their cattle. results are not advantageous in that regard. Most of the farmers interviewed were 51 years of age or older (≥51 years) and their source of income was in form of grants or pensions. Comparable findings by Scholtz et al. (2008) showed that rural/urban migration of the youth in search for greener pastures contributed to the higher proportion of rural farmers who were 60 years. This was also observed by Tada et al. (2012) who reported that young people usually go to urban areas to pursue their tertiary aspirations and secure better paying jobs. There were low cattle numbers per household herd and these findings are in agreement with Mapiye et al. (2009b) whose study revealed herd sizes of five to ten possibly due to unavailability of good quality rangelands. The unavailability of palatable and nutritious grazing land could Figure 8. Farmer’s response on tick-borne disease prevalence. 1668 Katiyatiya et al. (2014) Asian Australas. J. Anim. Sci. 27:1663-1670 Table 4. Association between demographic information and reasons for keeping specific cattle breeds Demographic Heat Milking Tick Meat quality Temperament Fertility factor tolerance ability tolerance Area NS NS NS * Gender ** NS NS NS NS NS Level of education NS NS NS * NS NS Employment NS NS NS * NS NS Traditionalism NS NS NS NS Knowledge on adaptation NS NS * NS Significant at * p≤0.05, p≤0.01, but NS not significant at p≥0.05. have contributed to the low numbers since the areas have different rainfall patterns which in turn influence vegetation growth (Mapiye et al., 2009a). There were more cows than heifers and this could have been due to the long calving intervals or mortality of calves causing low calf numbers on the other hand as observed in a study by Nqeno et al. (2011). Most of the farmers had at least one bull to use for breeding purposes though high oxen numbers were observed. These findings concurred with a study by Chimonyo et al. (1999) who mentioned that farmers tend to castrate their bulls in order to have oxen for draught power purposes. In contrast to a previous study by Tada et al. (2012) and Enkono et al. (2013) where cattle were mainly used for sales to obtain money, the farmers in the Eastern Cape Province considered milk as the major purpose for keeping cattle and on the other hand this finding was also reported by Musemwa et al. (2010). However, the farmers in the current study also kept cattle for ceremonies, meat, draught, manure, lobola and status quo as observed in similar studies by Mapiye et al. (2009a) and Nqeno et al. (2011). This indicated that there are variations from one farmer to another in the way they utilize cattle and their produce. Nguni and non-descript cattle were kept by the majority of the farmers as they reported that these were easily accessible and available animals in their communities. Several studies (Muchenje et al., 2008b) reported similar findings arguing that communal areas are characterized by harsh environments that require the raising of hardy breeds such as the Nguni. The non-descript cattle arose from the indiscriminate crossing of the Nguni with exotic (Bos taurus) breeds. The farmers did not regard the milking ability of the breeds as the major reason for keeping cattle. This was a contradiction since their general reason for cattle production was for milk purposes. Results from the study suggest that these breeds have good heat and tick tolerance abilities. Heat stress indicators perceived by the farmers showed that they had knowledge on heat regulation in cattle. Further reports were made that cattle usually graze in camps populated with canopy trees to provide shade as well and local dams for additional drinking water. In addition, some of the farmers provide additional drinking water in water points. Nienaber and Hahn (2007) postulated that shades aid in masking the animals from solar radiation. Farmers reported a higher prevalence of ticks during the summer season and these results were similar to the ones observed by Muchenje et al. (2008b) and Marufu et al. (2011) whose findings showed high tick prevalence in the rainy season. This has a negative impact on the productivity of the cattle leading to great economic losses. The farmers in the current study were concerned about the transmission of heartwater, redwater and gall sickness and similar findings in the Eastern Cape Province were highlighted by Table 5. Association between demographic information and effects of heats stress in cattle Demographic Excessive Loss of Reduced milk Reduced breeding Reduced actor panting weight production efficiency feed intake Area NS NS NS NS Gender NS NS NS NS NS Age NS NS NS NS NS Level of education NS NS NS NS NS Death Coat colour Hair length NS NS ** * NS NS NS NS NS * Significant at * p≤0.05, ** p≤0.01, but NS not significant at p≥0.05. Table 6. Association of demographic information and effects of ticks on cattle Demographic Reduced Disease Undesired Tick worry factor feed intake transmission meat quality Area NS * *** NS Gender * * NS NS Level of education NS NS ** NS Employment NS NS NS NS Significant at * p≤0.05, ** p≤0.01, but NS not significant at p≥0.05. Death Coat colour Hair length NS NS NS * * NS NS NS NS NS NS NS Katiyatiya et al. (2014) Asian Australas. J. Anim. Sci. 27:1663-1670 Mapiye et al. (2009a,b) and Nqeno et al. (2011). Hence, the farmers had resorted to the use of acaricides and ethnoveterinary medicines to control ticks and tick-borne diseases. The results are also comparable to findings by Ndebele et al. (2007) in Zimbabwe where 50% of the communal farmers used ethno-veterinary medicines and 90% made use of veterinary medicines provided by extension officers and government. For maximum efficiency, some used both control measures and dipped their cattle every fortnight in the dry season. Findings by Masika et al. (1997) also showed that farmers prefer to use ethno-veterinary medicines in addition to acaricides. This was because they perceived that ticks were now resistant to the acaricides provided by the state and they also wanted to completely eradicate tick problems in their cattle. Some of the farmers complained that the acaricides provided by the government were no longer as effective as the ones they used in the past years. The association of area with the perceptions on heat tolerance and fertility as reasons for keeping specific breeds may be due to the fact that, heat affects the productivity of cattle. Some areas experience higher environmental temperatures which may lead to stressful conditions when the heat lost by an animal is less than heat gained as postulated by Bernabucci et al. (2010). Hansen (2009) also found that heat stress affects fertility by disrupting spermatogenesis and oocyte development, oocyte maturation, early embryonic development, foetal and placental growth and lactation. Area was also associated with perceptions on excessive panting, death and coat colour. This was probably because animals tend to pant increasingly as a cooling mechanism and death results when the animal fails to release more heat than gained to the environment. The perceived findings that coat colour influences tick infestation in cattle were also reported by Bernabucci et al. (2010) who revealed that dark coat colours absorb more heat energy causing animals in areas with high ambient temperatures to be at risk. The ticks attach more often on dark coloured animals which make it difficult for predators to locate them. Studies by Ibelli et al. (2012) showed similar findings on coat characteristics and tick infestation. However, a study by Tada et al. (2013) revealed that farmers preferred certain coat colours based on culture, ceremonial significance and ritual activities. Gender was associated with the perceptions that nondescript and Nguni cattle are heat tolerant and that coat colour influence heat stress susceptibility in cattle. Most men also perceived that ticks cause tick worry and reduced feed intake in cattle. On the other hand, the level of education was associated with the perception that farmers chose specific cattle breeds because of their excellent meat quality attributes and that they had knowledge on cattle 1669 adaptation mechanisms under differing environmental conditions. The meat quality perception was similar to findings by Rani et al. (2013) who revealed that educated and employed people prefer lean meat for health reasons. This indicates that farmers know that the sustainable production of cattle has a contribution to the resulting beneficial meat quality attributes to people. Others perceived that hair lengths enhanced the heat tolerance ability of cattle while ticks can transmit tick-borne diseases. According to the farmers, some receive awareness and training from extension officers and this greatly contributed to their knowledge on cattle production and diseases as older farmers feared their animals were prone to death due to heat stress and ticks. CONCLUSION Results from the present study indicated that the most important reason for keeping cattle was for milk consumption. Nguni and non-descript cattle were reported to be the common breeds kept because of their heat resistance and tick tolerance abilities. The farmers perceived that ticks and heat stress can result in undesired meat quality, infertility and reduced weights of cattle. Coat colour and hair lengths were reported to have an influence on tick loads and heat stress in the summer seasons. The farmers commonly used acaricides and ethno-veterinary medicines to prevent tick infestations. Generally, the farmers had knowledge on cattle adaptation to heat stress and tick infestation in cattle. However, it is important to establish the tick load and heat stress attributes of Nguni cows by determining their coat characteristics, cortisol levels, temperature humidity index and haematological parameters. 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https://openalex.org/W4289076181	https://zenodo.org/records/3592703/files/Liberal-Studies-Journal-July-Dec-2018-111-121.pdf	English	null	HIV/AIDS, Security, and the United States of America	Zenodo (CERN European Organization for Nuclear Research)	2,018	cc-by	4,715	Abstract This paper debates on how pandemics like HIV/AIDS have become an important security concern for many countries and especially the United States of America. While the focus here, is on this particular epidemic, which has shaken international politics since the 1980s, it takes a more generic approach to what measures states undertake when it comes to protecting their interests, their jurisdictive geo-spatial spaces and the people governed by them. The humanitarian side of it is of course acknowledged, but along with that comes state interests, which transcend humanitarian beliefs and ethics. The relationship between humanitarian ethics and security has been kept so ambiguous and blurred in formal writings on the subject, that the various dimensions of security are often ignored by states. Often, under the pretext of economic growth and military expansion, individual wellbeing of the people being governed is equated merely with the state’s interest in productivity. The stronger states, in their efforts to alleviate the weaker ones, customarily patronize the latter, thereby creating a wider gap and disparity, leading to further dependence. Keywords: HIV/AIDS, Epidemic, Humanitarian, Security, USA. * The author is a Postdoctoral Fellow at the International Institute for Global Health, Kuala Lumpur, Malaysia, and Assistant Professor, Department of Political Science, Shaheed Bhagat Singh College, University of Delhi, India. ** Doctoral Candidate, CSRD, School Of Social Sciences, Jawaharlal Nehru University, New Delhi, India. Shantesh Kumar Singh* Mukund Upadhyay HIV/AIDS, Security, and the United States of America Doctoral Candidate, CSRD, School Of Social Sciences, Jawaharlal Nehru University, New Delhi, India. * The author is a Postdoctoral Fellow at the International Institute for Global Health, Kuala Lumpu Malaysia, and Assistant Professor, Department of Political Science, Shaheed Bhagat Sing College, University of Delhi, India. Introduction The idea of sovereignty is perhaps as old as the idea of the formation of the states itself. Respect for sovereignty and non-interference in each other’s administrative jurisdiction are the cardinal principles on which the states exist as independent entities and what they fundamentally demand of each other. Like mediaeval times, modern democratic nations pursue the same old strategy for survival and self-protection, but often, this is done in the guise of humanitarian Liberal Studies, Vol. 3, Issue 2, July–December 2018 260 and welfare measures, which are used as an excuse to impose, control, suppress, and finally to invade. When it comes to health, the need for security and humanity go hand in hand. No doubt, strategic protection from infectious diseases has necessitated global intervention but the definition of security is so ambiguous, that global actors have manipulated it to influence not only individual consciousness but the global consciousness as well. In this context, the relationship between HIV/AIDS and national security gains importance, the reason being its slow incubation period, history of incurability, its devastating impact on individuals, groups, communities, institutions and nations at large, and finally because of the fear of its global transmission. In the present-day scenario, international relations are no longer defined as the cat and mouse game of the past and with the development of human and state ethics, perhaps the idea of security has also changed; from the very obscurantist to a relatively progressive one. (In terms of comparison and not in the absolute sense of the words) This however did not happen in a vacuum, but through decades of intellectual and political discourses on the Public Health System. In the field of international relations, security is understood through the perspective of the state, while HIV/AIDS is looked at more through a political lens, where the intervention in its prevention and treatment is not entirely on humanitarian grounds, but largely supplemented by the idea that a pandemic can influence internal security too (McInnes, 2006), destroy labour productivity and hinder national growth.1 This has led to the unfortunate practice of quarantining weaker states by the more developed ones. The Approaches to Security and HIV/AIDS Constructivists take an objective view of military interventions and the power struggle exhibited by powerful states, but also focus on the subjective meaning of security as fabricated by states, societies, or individuals, based on their historical, cultural, or psychological understanding of intimidation and security. (Lo and Yuk, 2015) Recognising the importance of health to national security, states now include health as an integral component of military strategy. In the post-Cold War period, modern nation states have become increasingly concerned about the security issues relating to global public health, especially when it comes to addressing the spread and containment of infectious diseases. Hence, every approach to deal with an infectious disease being followed be it leprosy or HIV/AIDS, it would appear to become a security concern. What also compels critical attention is the way economic interests, like health interests, have merged with the larger security aspects. McInnes and Rushton (2011) argue that the concept of security has changed from a classical HIV/AIDS, Security, and the United States of America 261 military definition to a broader approach which includes the not so well-defined factors influencing the socio-political environment such as the physical environment, food, and migration, to name a few. To that extent modern approaches by states are confronted by the disquieting question: ‘What is not a security issue?’ Experts on the subject have said that even though health, as a human security issue, has a humanitarian flavour, it must connect to national security. McInnes (2006) claims that pandemics like HIV/AIDS are more easily transmitted in conflict prone areas and hence sensitive to military and national security. It would appear that we are living in an age of deception and contradictions where security is the mask under which health strategies too, find a convenient refuge. In her article entitled, ‘What contribution can International Relations make to the evolving global health agenda?’ Davies and Fidler mention two approaches towards public health in the international arena–the ‘statist’ approach and the global approach. The former mainly focuses on the security aspect where the health aspect is considered a mere strategy in a nation’s overall foreign and defence policy. They portray weak states as being largely dependent on stable governance for their good health and conclude that they are, by implication, poor protectors of health. The Approaches to Security and HIV/AIDS AIDS4; all further having a detrimental impact on the military services of the states. (Rushton 2001) DuPont (2001) points out that if the prevalence of HIV/ AIDS continues to expand further in East Asia, it will increase the poverty levels, intensify the problem of resource allocation, and thereby have an overall impact on the democratisation process of the regions. This will not only add to the national security concerns of individual states but it will also affect the collective national security. While these countries are suppressed under the ruthless arms of poverty, poor public health attracts big corporates and governments from developed nations, (DuPont, IBID) who, as a political strategy, offer economic benefits in the guise of their securitization and humanitarian policies. (DuPont, 2001; Fidler, 2004; Rushton, 2000). The Approaches to Security and HIV/AIDS That is why the statist approach empowers states to put forward their public health agenda by securitization of the area commonly called ‘high politics.’ (Davies, 2001; Fidler, 2004) Though the globalist approach acknowledges various aspects of health concerns in the everyday lives of the people and is sensitive towards the individual’s health needs and expects the state to cater to those needs in a humanitarian manner, it does not necessarily consider the state as being vital to the needs of individuals. While the global approach does not entirely reject the importance of national security, it does not concur with the exclusivity of the ‘statist’ approach of securitization of states. In the case of infectious diseases, the globalists analyse it in their own right and argue that regardless of the fact of who is infected, it is important that intervention be carried out in the name of humanity. The statist, on the other hand, as Davies points out, is focussed on protecting a specific group or population to whom it is accountable. Developed nations strongly believe that HIV/AIDS is posing a major threat to the global social environment and its psycho-social well being, which is detrimental to the stability of states or regions.2 They have coined the term ‘hollowing out’ to describe the socio-economic impact of effective production, caused by illnesses and premature death (Vieira 2007) on the state institutions by the loss of life. This, they claim is leading to deviant behaviour in the youth3 and the increasing problem of orphaned children of parents dying from HIV/ Liberal Studies, Vol. 3, Issue 2, July–December 2018 262 AIDS4; all further having a detrimental impact on the military services of the states. (Rushton 2001) DuPont (2001) points out that if the prevalence of HIV/ AIDS continues to expand further in East Asia, it will increase the poverty levels, intensify the problem of resource allocation, and thereby have an overall impact on the democratisation process of the regions. This will not only add to the national security concerns of individual states but it will also affect the collective national security. While these countries are suppressed under the ruthless arms of poverty, poor public health attracts big corporates and governments from developed nations, (DuPont, IBID) who, as a political strategy, offer economic benefits in the guise of their securitization and humanitarian policies. (DuPont, 2001; Fidler, 2004; Rushton, 2000). The Changing Nature of State Approach to Security and HIV/AIDS: A Tale of American Interest The HIV/AIDS pandemic has not only affected individuals, but, over the years, has impacted the psycho-social, economic and national development of many potential societies, leaving large parts of the world in a post-traumatic condition. Unlike other pandemics, when we talk about security in the context of HIV/AIDS, we must take into account various aspects of individual, family, community, economic, military, and global health security, which influence individual behaviour, institutional and structural orientation and overall societal wellbeing. Security being associated with HIV/AIDS has seen the emergence of new policies influenced by health and medical experts, leading to a wider process of medicalization. (Elbe, 2011). Among the security concerns associated with HIV/AIDS, the technical aspect of security in its different perspectives has been discussed in many scholarly dissertations. Often the term security is associated with national and global security (Cecchine and Moore, 2006) almost completely, or at least partially, ignoring the most fundamental aspect of security, which should be family and societal security. This is so, mainly owing to the fact that the major focus has been appropriated by military and institutional concerns, which are considered as the essential foundation of national security by world leaders. The pandemic is no longer merely a humanitarian crisis, but a security crisis that emerges from the larger consciousness of the state to protect itself and its institutional interests.5 In the process, personal security, which is related to human life span and productivity and forms the basis of individual wellbeing, gets increasingly threatened by a pandemic like HIV/AIDS. The 108th Congress of the United States of America, in its capitalist mode of comprehension of public health, and particularly infectious diseases, pointed out that HIV/AIDS can be a major threat HIV/AIDS, Security, and the United States of America 263 to national productivity, which could lead to losses in the growing business of the country. The Congress, although it acknowledged the importance of individual wellbeing, considered it from the perspective of labour and productivity, and did not hesitate in pointing out that the pandemic is a threat to international business and security, not forgetting to mention its effect on the American military under the UN peace keeping forces deployed in war-torn underdeveloped countries of Africa and Asia. The Changing Nature of State Approach to Security and HIV/AIDS: A Tale of American Interest Section 2, Article 10 of the Congress mentions how the American military under the UN peace keeping force has a greater likelihood of getting infected in those environments in which it operates. The idea here is to analyse how the United States’ concept of security is confined to the economic boundaries defined by capitalist interests and American internationalism. Authors like Fieldbaum (2006) and others have pointed out the hypocritical nature of the United States and its duality in failing to identify non-communicable diseases that are a threat to national security. As mentioned earlier, developed nations like the United States have used military intervention for their own economic expansion, but in the guise of humanitarian concern. To address the prevention and treatment of HIV/AIDS the emphasis has shifted from the Western Phalian approach which was based on the principle of non- interference in the sovereignty of nations and respect for self-determination to the Post-Western Phalian approach wherein aspects of human rights have been given prime importance. (Fidler, 2004) The United States of America came a long way from being a nation which had neglected the HIV/AIDS pandemic during the Reagan administration to a more progressive humanitarian Obama administration. But again, it has become very apathetic and regressive under the Trump administration. Over the years, the idea of America’s National security as a state strategy has been the focus of attention and has taken precedence over the humanitarian and ethical path. In this however, the American ideals, interests and internationalism had always been of prime concern, especially the protection of American military from infection during its operations in Africa and Asia. Though the US was involved in securing itself from the HIV/AIDS pandemic it was only during the 1990s (Rushton, 2000; Fidler, 2004; Vieira, 2007) that the HIV/AIDS-security linkage became apparent in Washington policy circles. Some of those involved in pushing forward the case complained that the Clinton administration was somewhat slow on the uptake. Though the argument was gaining ground, such a pandemic concerning global public health did not receive the kind of attention it demanded from a global power like the United States of America. In fact, the regressive mind-set of the American administration was only too evident. In 1982, in an interview with the journalist and radio host Liberal Studies, Vol. The Changing Nature of State Approach to Security and HIV/AIDS: A Tale of American Interest 3, Issue 2, July–December 2018 264 Lester Kinsolving, Larry Speakes, Press Secretary to Ronald Reagan, could not resist making fun of the HIV/AIDS pandemic as a ‘gay disease’ and mocking those suffering from it. (Lopez, 2016) Even after that, there was very little effort made by the Reagan administration to address the HIV/AIDS problem and it was only years later, in 1990, that the Ryan White Care Act6 was launched in America during President George H. W. Bush’s tenure. Recognising the serious threat posed by HIV/AIDS, the Clinton administration (1993-2001) increased the funds for the prevention and treatment of HIV/AIDS and also put in place other measures to combat the pandemic. America’s idea of security as a component of HIV/AIDS prevention and treatment has its genesis in the United States Ambassador to the United Nations, Richard Holbrooke’s visit to Africa in 1999 to get an overview of the AIDS outbreak in that country.7 It was Holbrooke who redefined the HIV/AIDS pandemic as world-wide as well as an internal stability threat, though most give President Clinton credit for it. It was his seminal observation of the condition of the US military in the UN peace keeping forces in Cambodia8 and Africa and his continuous persuasion of the Secretary-General of the United Nations, Kofi Annan, that led to the UN Security Council Resolution 13089 being passed in July 2000 and the pandemic being included as a threat to international peace and security. (Vieira, 2007; Cecchine and Moore, 2006). During this period, the situation in Africa had grown so serious that there were more deaths from HIV/AIDS than from civil conflicts. In December 2004, the UN Secretary-General’s High-Level Panel Report on Threats, Challenges and Change, highlighted the need for immediate intervention to deal with the pandemic and to protect state and societies through proper research and planning and action taken to reduce the impact of the endemic in the long run. (Tony Barnett and Gwyn Prins, 2006) This was an event of paramount importance because, for the first time, the popular notion of public health and development was brought under the ambit of international security. (Stefan Elbe, 2006). US Perspectives on HIV/AIDS and Security Although the Security Council had started monitoring the securitization process, its efforts were half-hearted and non-committal. Rushton (2000) argues that discourses around the securitization of HIV/AIDS have been far less successful than it is often supposed. Meanwhile, though the security threats posed by the African nations on the United States vis-a-vis, HIV/AIDS had been widely debated, from the period of the Clinton administration, there was a gradual shift in US thinking, with attention being diverted from Africa to countries like India, Russia and China where the US saw greater opportunities HIV/AIDS, Security, and the United States of America 265 for involvement to further their economic interests. It is no secret that the US designs its strategies and re-defines its core and peripherals based on its own interests, and though sub-Saharan Africa was most important to the national security debate it was ultimately peripheral. (Fidler, 2003). The United States has repeatedly, time and again pointed out that infectious diseases endanger its citizens at home and abroad and also puts the US armed forces deployed in different missions, overseas, at risk. Infectious diseases exacerbate social and political instability in regions where the US has been showing interest. While infectious diseases are addressed in terms of interventions, one cannot say the same for non-communicable diseases as far as US interventions are concerned. Such strategic focus by the US has isolated important global health problems that exhibit high morbidity and mortality. The lack of interest shown to non-communicable diseases by powerful states like the US in developing countries only goes to prove that non-communicable diseases do not meet their strategic interests and hence the politics hidden in the guise of security and humanitarianism becomes quite evident. During the initial years of the detection of HIV/AIDS, it was relegated to the category of public health and development in the US and was not given due importance. Though the US Central Intelligence Agency endeavoured to link HIV/AIDS with security in the 1990s, it could not get the attention it required until 2000 in the form of the 1308 resolution. (Fidler, 2004; Cecchine, Moore, 2006) A National Intelligence Council report entitled, ‘The Global Infectious Disease Threat and its Implications for the United States’, which supported the Clinton Administration’s portrayal of HIV/AIDS as a global security threat, analysed the devastating internal impact caused by the HIV/AIDS pandemic. Concluding Remarks and Concerns The idea of security has always been a major concern for countries like the United States. On one hand, the United States has extended support to developing countries through its different policies, but on the other, it has also engaged in manipulating, re-constructing and re-orienting the idea of security by putting too much focus on the technical aspects pertaining to national security, and through it all, has missed the essentials of human rights and humanitarianism. The United States has not been able to address the global HIV/AIDS pandemic in recent years in the way it has been expected to, and American Internationalism, hidden in the sympathetic nature portrayed by Bush or the empathetic nature of Obama is no less harmful than the outwardly ignorant attitude of President Reagan in the early 1980s and the very apathetic nature of President Trump today. The intention here is not to discredit or belittle the efforts of successive American governments, but to better appreciate the fact that the concept of security should not remain confined to the American notion of security, which focuses on mere labour productivity, capital expansion and military security. The idea of security has always been a major concern for countries like the United States. On one hand, the United States has extended support to developing countries through its different policies, but on the other, it has also engaged in manipulating, re-constructing and re-orienting the idea of security by putting too much focus on the technical aspects pertaining to national security, and through it all, has missed the essentials of human rights and humanitarianism. Along with the complexities of imposing the responsibility on developing nations, the United States has also been discriminatory in its approach within its own boundary. Although this is not an important aspect in this paper, it is nonetheless a matter of concern, which reflects the plight faced by people of developing countries when it comes to addressing the problem of the HIV/AIDS pandemic. The ethical question related to the securitization of HIV/AIDS is well mentioned by Elbe (2011) who traces the cases of discrimination and marginalization faced by countries of suspect, as in the case with Haitians in the United States, who are denied housing or other basis facilities of life and sometimes even dismissed from jobs. US Perspectives on HIV/AIDS and Security (Vieira, 2007) Developing nations, immigrants and retired US personnel were held responsible for its spread and the report claimed that it not only had a tremendous impact on internal security and the military, but also on the very socio-political fabric of the United States of America. (John C. Gannon, 2000). However, scholars like Susan Peterson argued that such a portrayal of the pandemic was exaggerated and maybe slightly overestimated, and that the security implications had insufficient arguments to support it, which might only fuel suspicion, rivalry and conflict between states, thereby triggering other security concerns. She advocated a more progressive approach in dealing with the issue, through better multi-national co-operation. Peterson further argued that responding to pandemics like HIV/AIDS only through the perspective of national security would, in the long run, discourage nations and eliminate the chance of states addressing public health issues in their own right. The garrisoning of states over human rights might provide them the opportunity to be vigilant, Liberal Studies, Vol. 3, Issue 2, July–December 2018 266 but they could abdicate from the moral responsibility that they should have towards the weak and the vulnerable. Peterson, in this regard, argues that the nation-states should strengthen their capacity to actually face the pandemic rather than securitizing states against it. States should come up with interventional plans to deal with the pandemic for what it is and recognize the unprecedented tragedy it could bring and endeavour to build a network of international humanitarian assistance to solve the problem of HIV/AIDS. (Susan Peterson, 2002). Notes 1. United Nations special session on HIV/AIDS, June 2001. http://www.un.org/ga/aids/ungassfactsheets/html/fssecurity_en.htm, and the One Hundred Tenth Congress of the United States of America held in the City of Washington on Thursday, 03 January 2008, https://www pepfar gov/documents/organization/108294 pdf https://www.pepfar.gov/documents/organization/108294.pdf. 2. 108 Congress of the United States of America held in Washington DC, 2003, https: www.congress.gov/bill/108th-congress/house-bill/1298. 3. Adewole, “Public health, national security interlinked”, New Telegraph, September 2018, https://newtelegraphonline.com/2018/09/public-health-national-security-interlinked- adewole/. 3. Adewole, “Public health, national security interlinked”, New Telegraph, September 2018, https://newtelegraphonline.com/2018/09/public-health-national-security-interlinked- adewole/. 4. AIDS as a security threat, United Nations Special Session on HIV/AIDS, Global Crisis- Global Action, June 2001, New York, http://www.un.org/ga/aids/ungassfactsheets/html/ fssecurity en.htm. 4. AIDS as a security threat, United Nations Special Session on HIV/AIDS, Global Crisis- Global Action, June 2001, New York, http://www.un.org/ga/aids/ungassfactsheets/html/ fssecurity en.htm. 5. Speech by US Vice President Al Gore during the UN Security Council meeting, 10 January 2000. 6. About the Ryan White HIV/AIDS Program, date last reviewed October 2016, https:// hab.hrsa.gov/about-ryan-white-hivaids-program/about-ryan-white-hivaids-program. 7. Interview: Richard Holbrooke, The Frontline, 30 May 2006, https://www.pbs.org/wgbh/pages/frontline/aids/interviews/holbrooke.html. 8. Ibid. 7. Interview: Richard Holbrooke, The Frontline, 30 May 2006, https://www.pbs.org/wgbh/pages/frontline/aids/interviews/holbrooke.html. 8 Ibid 9. UN Security Council Resolution 1308 (2000) on the Responsibility of the Security Council in the Maintenance of International Peace and Security: HIV/AIDS and International Peace- keeping Operations Security Council Distr.: General 17 July 2000, http://www.unaids.org/sites/default/files/sub_landing/files/20000717 un scresolution _1308 en.pdf. 10. Ibid. 10. Ibid. Concluding Remarks and Concerns Similar was the fate of Africans in Europe who were denied basic rights because of the fear of them being HIV/AIDS carriers.10 The practice of discrimination followed by countries like the United States, who, on the one hand, portray themselves as torch bearers of the humanitarian philosophy, and on the other, discriminate against migrants and settlers infected by HIV is HIV/AIDS, Security, and the United States of America 267 definitely a form of double standard, a paradox and oxymoronic in the American context. It is contradictory because it is a well-established fact that HIV/AIDS has definite criteria for infection and people cannot be infected through the physical environment and, therefore, discriminating against certain ethnic groups, for example and not providing them houses, and other basic amenities is an act which reflects racist perceptions associated with HIV/AIDS. This paper is a part of the ongoing ICSSR sponsored research project entitled “A Critical Study of the United States fight Against HIV/AIDS in India”. We acknowledge and thank all those who helped us during the research project. We declare that there is no conflict of interest. References 1. T Barnett and G. Prins, “HIV / AIDS and Security: Fact, Fiction and Evidence: A Report to UNAIDS”, International Affairs (Royal Institute of International Affairs 1944-), Vol. 82, No. 2, HIV/AIDS-Special Issue, 2006, pp. 359-368, Oxford University Press on behalf of the Royal Institute of International Affairs, Accessible at: http://www.jstor.org/stable/ 3569426, accessed on 22 November 2017. Liberal Studies, Vol. 3, Issue 2, July–December 2018 268 2. G. W. Brown and R. Labonté, “Globalization and its methodological discontents: Contextualizing globalization through the study of HIV/AIDS”, Globalization and Health, 7, 29, 2011, http://doi.org/10.1186/1744-8603-7-29. 3. N.G. Cecchine and M. Moore, “Technical Report on Infectious Disease and National Security Strategic Information”, prepared for the Office of the Secretary of Defence Approved for public release, National Defence Research Institute, Rand Corporation, 2006. https://www.rand.org/content/dam/rand/pubs/technical reports/2006/RAND TR405.pdf. 4. S.E. Davies, “What contribution can International Relations make to the evolving global health agenda, International Affairs”, Royal Institute of International Affairs, Vol. 86, No. 5, 2010, pp. 1167-1190, Oxford University Press on behalf of the Royal Institute of International Affairs, http://www.jstor.org/stable/40865133. 5. A. DuPont, East Asia Imperilled: Transnational Challenges to Security, Cambridge, Cambridge University Press, 2001. 6. Stefan Elbe, “Should HIV/AIDS Be Securitized? The Ethical Dilemmas of Linking HIV/ AIDS and Security”, International Studies Quarterly, Vol. 50, No. 1, 2006, pp. 119-144, Wiley on behalf of The International Studies Association, http://www.jstor.org/stable/ 3693554. 7. Stefan Elbe, “Pandemics on the Radar Screen: Health Security, Infectious Disease and the Medicalisation of Insecurity”, Political Studies, 59, 4, 2011, pp. 848-866. 8. H. Feldbaum, K. Lee and P. Patel, “The National Security Implications of HIV/AIDS”, PLoS Med 3(6): e171, 2006, https://doi.org/10.1371/journal.pmed.0030171. 9. D. P. Fidler, “Racism or Realpolitik – U.S. Foreign Policy and the HIV/AIDS Catastrophe in Sub-Saharan Africa”, Gender Race & Justice, 97, 2003, p. 148. 10. D.P. Fidler, “Fighting the Axis of Illness: HIV/AIDS, Human Rights, and U.S. Foreign Policy”, Maurer Faculty, Paper 400, 2004, http://www.repository.law.indiana.edu/facpub/ 400. 11. John C. Gannon, “The Global Infectious Disease Threat and Its Implications for the United States”, NIE 99-17D, January 2000, https://fas.org/irp/threat/nie99-17d.htm. 12. G. Lopez, “The Reagan administration’s unbelievable response to the HIV/AIDS epidemic”, Vox, 2016, https://www.vox.com/2015/12/1/9828348/ronald-reagan-hiv-aids. 13. C. McInnes and S. Rushton, “HIV/AIDS and securitization theory”, European Journal of International Relations, 19(1), 2011, pp. 115–138, http://journals.sagepub.com/doi/pdf/ 10.1177/1354066111425258. 14. C. McInnes, “HIV/AIDS and security”, European Journal of International Relations¸ 2006, https://doi.org/10.1111/j.1468-2346.2006.00533.x. 15. Susan Peterson, “Epidemic Disease and National Security”, Journal of Security Studies, vol. 18. A.M. Vieira, “The Securitization of the HIV/AIDS Epidemic as a Norm: A Contribution to Constructivist Scholarship on the Emergence and Diffusion of International Norms”, Brazilian Political Science Review, vol. 2, Rio De Jenerio, 2007, http://socialsciences. scielo.org/pdf/s_bpsr/v2nse/a05v2nse.pdf. 19. C Yuk and P. Lo, HIV/AIDS in China and India Governing Health Security, Palgrave Macmillan Publication, New York, 2015. References 12, no. 2, 2002, pp. 43-81. 16. S. Rushton, “AIDS and international security in the United Nations System”, Health Policy and Planning, Vol. 25, Issue 6, 1 November 2010, pp. 495–504, https://doi.org/10.1093/ heapol/czq051. 17. Tony Barnett and Gwyn Prins, “HIV / AIDS and Security: Fact, Fiction and Evidence: A Report to UNAIDS”, International Affairs (Royal Institute of International Affairs 1944), Vol. 82, No. 2, HIV/AIDS-Special Issue (March 2006), pp. 359-368, published by: Oxford University Press on behalf of the Royal Institute of International Affairs, http:// www.jstor.org/stable/3569426. HIV/AIDS, Security, and the United States of America 269
https://openalex.org/W4224094880	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0266753&type=printable	English	null	Integrated use of phosphorus, farmyard manure and biofertilizer improves the yield and phosphorus uptake of black gram in silt loam soil	PloS one	2,022	cc-by	7,839	RESEARCH ARTICLE Integrated use of phosphorus, farmyard manure and biofertilizer improves the yield and phosphorus uptake of black gram in silt loam soil Jagdish SinghID1, Rajan Bhatt2, Dhaliwal S. S.3, B. S. Dhillon1, Asma A. Al-Huqail4, Alanoud AlfaghamID4, Manzer H. SiddiquiID4, Hayssam M. AliID4, Faheema Khan4, Ritesh KumarID5 1 Punjab Agricultural University Regional Research Station, Gurdaspur, Punjab, India, 2 Punjab Agricultural University Regional Research Station, Kapurthala, Punjab, India, 3 Department of Soil Science, Punjab Agricultural University Ludhiana, Punjab, India, 4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia, 5 Department of Agronomy, Kansas State University, Manhattan, KS, United States of America * jagdishsingh@pau.edu (JS); mhsiddiqui@ksu.edu.sa (MHS) RESEARCH ARTICLE Integrated use of phosphorus, farmyard manure and biofertilizer improves the yield and phosphorus uptake of black gram in silt loam soil Jagdish SinghID1, Rajan Bhatt2, Dhaliwal S. S.3, B. S. Dhillon1, Asma A. Al-Huqail4, Alanoud AlfaghamID4, Manzer H. SiddiquiID4, Hayssam M. AliID4, Faheema Khan4, Ritesh KumarID5 1 Punjab Agricultural University Regional Research Station, Gurdaspur, Punjab, India, 2 Punjab Agricultural University Regional Research Station, Kapurthala, Punjab, India, 3 Department of Soil Science, Punjab Agricultural University Ludhiana, Punjab, India, 4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia, 5 Department of Agronomy, Kansas State University, Manhattan, KS, United States of America * jagdishsingh@pau.edu (JS); mhsiddiqui@ksu.edu.sa (MHS) myard s the yield PLOS ONE RESEARCH ARTICLE Jagdish SinghID1, Rajan Bhatt2, Dhaliwal S. S.3, B. S. Dhillon1, Asma A. Al-Huqail4, Alanoud AlfaghamID4, Manzer H. SiddiquiID4, Hayssam M. AliID4, Faheema Khan4, Ritesh KumarID5 1 Punjab Agricultural University Regional Research Station, Gurdaspur, Punjab, India, 2 Punjab Agricultural University Regional Research Station, Kapurthala, Punjab, India, 3 Department of Soil Science, Punjab Agricultural University Ludhiana, Punjab, India, 4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia, 5 Department of Agronomy, Kansas State University, Manhattan, KS, United States of America 1 Punjab Agricultural University Regional Research Station, Gurdaspur, Punjab, India, 2 Punjab Agricultural University Regional Research Station, Kapurthala, Punjab, India, 3 Department of Soil Science, Punjab Agricultural University Ludhiana, Punjab, India, 4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia, 5 Department of Agronomy, Kansas State University, Manhattan, KS, United States of America 1 Punjab Agricultural University Regional Research Station, Gurdaspur, Punjab, India, 2 Punjab Agricultural University Regional Research Station, Kapurthala, Punjab, India, 3 Department of Soil Science, Punjab Agricultural University Ludhiana, Punjab, India, 4 Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia, 5 Department of Agronomy, Kansas State University, Manhattan, KS, United States of America a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 * jagdishsingh@pau.edu (JS); mhsiddiqui@ksu.edu.sa (MHS) * jagdishsingh@pau.edu (JS); mhsiddiqui@ksu.edu.sa (MHS) Editor: Shah Fahad, The University of Haripur, PAKISTAN PAKISTAN Received: February 27, 2022 Accepted: March 27, 2022 Published: April 14, 2022 Copyright: © 2022 Singh 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. Received: February 27, 2022 Accepted: March 27, 2022 Published: April 14, 2022 Copyright: © 2022 Singh 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. Abstract An experiment on the use of farmyard manure and biofertilizer along with application of chemical phosphorus was conducted to assess the impact of differential doses of phospho- rus, farmyard manure and consortium biofertilizer application on the development, yield and phosphorus uptake during the year 2018 and 2019. The impact of different treatments was recorded on the plant height, dry matter partition, yield and yield attributes, phosphorus uptake and soil phosphorus availability using standard methods. The data revealed signifi- cant improvement in yield, yield attributes, phosphorus uptake and soil phosphorus avail- ability. The integration of farmyard manure and biofertilizer with 60 kg ha-1 SSP (single superphosphate) has improved the black gram yield by 7.4% and 3.28% respectively over the use of 60 SSP alone. The phosphorus uptake in black gram with application of Farm yard manure and biofertilizer along with 60 kg ha-1 SSP has improved the uptake by 7.18% and 2.51% respectively over the use of 60 kg ha-1 SSP alone. The results highlight the need of integrated application of farm yard manure, biofertilizer for sustainable production of black gram in the region. OPEN ACCESS Citation: Singh J, Bhatt R, S. DS, Dhillon BS, Al- Huqail AA, Alfagham A, et al. (2022) Integrated use of phosphorus, farmyard manure and biofertilizer improves the yield and phosphorus uptake of black gram in silt loam soil. PLoS ONE 17(4): e0266753. https://doi.org/10.1371/journal.pone.0266753 Editor: Shah Fahad, The University of Haripur, PAKISTAN 1. Introduction Black gram (Vigna mungo L) is a leguminous short-season crop that is one of India’s most sig- nificant pulse crops. Black gramme seeds contain 17 to 34 percent protein, making them a cost-effective source of protein. [1]. The black gram was cultivated over 3.51 million ha area and production of about 2.08 million tons, with 593 kg ha-1 average productivity in India in the year 2017–18 [2]. Phosphorus is a primary macronutrient, essential for plant growth and development. The earth crust contains about 0.1% phosphorus, where most of this is present either in insoluble or unavailable form to plants. The low availability of phosphorus in the soil PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 1 / 10 PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil limits about 40% of crop production in cultivated areas around the globe [3]. The problem of P unavailability further aggravates as the added P fertilizers undergo fixation at exchange com- plexes within the soil make it unavailable to plants [4]. Phosphorus is an essential component of nucleic acid, phospholipids, protein, coenzyme, and the energy cycle, and plays a significant role in plant growth, root development, nodula- tion, nitrogen fixation, flowering, and fruiting [5–7]. The addition of phosphorus improves the grain yield of legumes crops such as Phaseolus vulgaris [8], Glycine max [9], Vicia faba [10], Cicer arietinum [11], Vigna unguiculata [12], etc. The excessive use of phosphatic fertilizers has been associated with soil and water pollution [6], i.e eutrophication of lakes and water bodies. The integrated use of organic manures and chemical fertilizers in crop production has proved to be helpful in maintaining higher crop productivity and sustainability in crop pro- duction [13]. The organic materials are known for their potential to act as an alternative source of multiple nutrients and their capability to improve the soil characteristics such as bulk den- sity, water holding capacity, soil microbial activities, soil structure and infiltration rate, while reduces soil compaction and soil erosion [14, 15]. Therefore, application of phosphorus with organic manure and bio fertilizers may help in compensating the supply shortage and rising price of chemical fertilizers. Many studies have concluded that crops utilize only 15–20% of the applied phosphorus [16, 17] and remaining get fixed in soil or converted to the form not readily available to the crops [18]. 2.1 The soil and the experimental site The investigation was carried out during the summer season of the year 2018 and 2019 to access the impact of unified phosphorus management on the growth, yield and phosphorus uptake in black gram. The field experiment was initiated at the farm of Punjab Agricultural University Regional Research Station, Gurdaspur, India, situated at 32.05˚ N and 75.43˚ E at an elevation of 225 m above mean sea level. The soil of the experiment site is characterized as the silt loam in texture with bulk density 1.49 Mg m-3, pH 7.6 (1:2 soil: water ratio), electrical conductivity of 0.20 dSm-1, 0.23% organic carbon, 19.0 kg ha-1 available phosphorus, 118.2 kg ha-1 available potassium. The experimental site is characterized as subtropical and semi-arid climate and receives about 700 mm rainfall annually. The weather during the study period is characterized as hot and dry summers (April-June), where temperature ranges from 16–42˚ C. The mean maxi- mum temperature recorded is as high as about 42˚ C in the May- June months. 1. Introduction Biofertilizers (Azotobacter, Azospirillum, Rhizobium, phos- phobacteria and VAM fungi) are proving to be cost-effective alternative to expensive fertilizers, supplement nitrogen and phosphorus fertilizers by fixing environmental nitrogen, converting insoluble phosphorus from the soil into soluble form, enhances soil nutrient availability to the plants [19] and improves crop yields. Integrated approach of nutrient management may derive maximum benefits from organic and inorganic fertilizers in an integrated manner and helps in maintaining optimum soil fertil- ity and crop productivity [20]. Hence, the experiments under consideration were undertaken to access the response of black gram to differential doses of phosphorus, FYM and consortium biofertilizer application. 2.3 Crop management The sowing of black gram cultivar Mash-1008 was done in the first fortnight of the march dur- ing the year 2018 and 2019. The sown of black gram was done with row spacing of 22.5 cm with seed rate of 50 kg ha-1. Pendimethlin @ 2.5 liter per hectare was sprayed after sowing to check the weeds. All the fertilizers application, weed control, insect-pest control, irrigation and other cultural practices were done as per the PAU package of practices for rabi crops. 2.2 Treatments and experimental design Current investigation comprised of eight treatments which laid out in randomized complete block design with three replicates during both the years. The treatments include T0-control, PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 2 / 10 PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil T1-60 kg ha-1 SSP, T2- 2.5 t ha-1farmyard manure (FYM), T3-2.5 t ha-1 FYM + 60 kg ha-1 SSP, T4-2.5 t ha-1 FYM + 30 kg ha-1 SSP, T5-consortium biofertilizer, T6-Consortium+60 kg ha-1 SSP and T7-Consortium+30 kg ha-1 SSP. The whole of the nitrogen was broadcasted at the time of sowing using urea (46% N) and phosphatic fertilizer was used as per treatments through single superphosphate (16% P2O5). Each replication plot was 2.6 m wide x 3.0 m long. The well rotten farmyard manure (FYM) was added to surface soil and mixed before sowing as per treatment. The FYM had 0.58% N, 0.25% P and 0.57% K. The consortium biofertilizer con- sist of Phosphorus Solubilizing Bacteria, Azotobacter and Plant Growth Promoting Rhizobac- teia. The powdered consortium biofertilizer was moisten and uniformly allocated over black gram seeds. The consortium biofertilizer coated seeds were air dried under shade before sowing. 2.4 Soil and plant observations In each plot, five plants were chosen at random and tagged to record observations on plant height, branches per plant, number of pods per plant, and number of seeds per pod. Dry mat- ter partitioning was performed on a representative plant from each plot, with leaf, stem, and roots dried separately. The roots were excavated by inserting iron pipe (6 inches diameter) into the soil keeping the plant base at the centre of pipe. The soil thus excavated was washed over the wire mesh under running water and roots were separated. The leaf, stem and roots were dried in an oven at 60˚C till the constant weight was achieved. The grain yield and straw yield were recorded on whole plot basis and converted into t/ha. The thousand grain weight at harvesting was also measured. Using a colorimeter, the phosphorus content of plant tissue was measured using the vanado-molybdate method [21]. Phosphorus uptake was calculated by multiplication of P con- tent (%) in grain with the grain yield (kg ha-1). 2.5 Analytical statistics The data from the plants were subjected to an analysis of variance (ANOVA) using the GLM procedure in SAS 9.1 statistical software (SAS, California). The significance of differences in means was tested with the Duncan Multiple Range Test. PLOS ONE No of pod Grain yield (t/ha) Biomass yield (t/ha) Thousand grain weight (g) Grain P content (%) T0-control, 18.6c 3.54d 16.51d 37.34b 0.207a T1-60 kg SSP, 25.9b 4.57ab 19.73ab 39.02a 0.215a T2- farmyard manure (FYM) 2.5 t/ha, 25.9b 4.24bc 18.01c 38.14ab 0.211a T3-2.5 t ha-1 FYM+60 kg SSP 29.7a 4.91a 20.99a 39.0a 0.215a T4-2.5 t ha-1 FYM + 30 kg SSP 26.5b 4.55ab 19.08bc 38.12ab 0.211a T5-consortium biofertilizer 21.5c 3.89cd 17.94c 37.92ab 0.209a T6-consortium biofertilizer +60 kg SSP 28.9ab 4.72a 20.39ab 38.7ab 0.213a T7-consortium biofertilizer +30 kg SSP 25.7b 4.12c 18.32c 37.78ab 0.209a CV 9.57 6.67 5.53 3.10 2.78 Table 1. Effect of phosphorus levels, farmyard manure and biofertilizer on plant height and dry matter accumulation. Phosphorus and soil physical environment. The accessibility of phosphorus also improves the utilization of N and K in the plant system. As phosphorus is involved in the energy cycle of plant in addition to its involvement in protein and lipids in the plant system, that help in improving plant growth. Phosphorus and soil physical environment. The accessibility of phosphorus also improves the utilization of N and K in the plant system. As phosphorus is involved in the energy cycle of plant in addition to its involvement in protein and lipids in the plant system, that help in improving plant growth. The data on the root dry matter accumulation in various treatments revealed that the early root growth of black gram at 35 DAS was significantly affected by the different combinations of source of phosphorus, FYM and biofertilizers (Table 2). The root dry mass at 35 DAS (days after sowing) was higher in the treatments where chemical fertilizer were integrated with the organic and biofertilizers such as T7 and T4 as compared to application biofertilizer, organic manure and chemical fertilizer alone. The root dry matter at the harvesting stage was signifi- cantly higher in the treatment T3 where FYM was applied in addition to the full dose of phos- phorus followed by use of biofertilizer and chemical fertilizers. Choudhary et al., [22] also reported beneficial effects of biofertilizer and organic manure on root growth and develop- ment. The recent studies had showed that phosphorus application enhanced root system resulting in better soil-root contact and finally higher P uptake and absorption of mineral nutrients [23]. d and yield attributes under different phosphorus levels, farmyard manure, and biofertilizer treatments. Table 2. PLOS ONE Treatments Plant height (cm) Root dry matter Leaves dry matter Branches/stem dry matter 30 DAS At harvesting 30 DAS At harvesting 30 DAS At harvesting 30 DAS At harvesting T0-control, 15.8c 26.1d 0.39g 2.94c 1.23g 5.31e 0.84c 4.43g T1-60 kg SSP, 17.3c 30.7abc 0.53d 3.91b 1.46cd 8.31bc 0.91bc 9.54b T2- farmyard Manure (FYM) 2.5 t/ha, 17.7b 29.5bc 0.43f 3.34c 1.60a 7.86c 0.93ab 5.67f T3-2.5 t ha-1 FYM+60 kg SSP 18.8a 32.0a 0.52e 4.85a 1.44de 9.26a 0.93ab 10.04a T4-2.5 t ha-1 FYM + 30 kg SSP 17.4b 30.7abc 0.65b 4.31b 1.53b 8.64abc 1.00a 8.33d T5-consortium biofertilizer 16.4c 28.7c 0.57c 3.89b 1.50bc 6.95d 0.89b 5.31f T6-consortium biofertilizer +60 kg SSP 18.5a 31.3ab 0.37h 4.29b 1.40ef 8.79ab 0.91bc 9.01c T7-consortium biofertilizer +30 kg SSP 17.5b 30.1abc 0.80a 4.03b 1.37f 8.18bc 0.84c 7.22e CV 3.22 5.68 5.57 8.30 3.20 7.96 6.28 5.30 Pr>F (Treatment) <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Pr>F (Year Treatment) 0.92 0.24 0.97 0.15 0.08 0.07 0.01 0.19 Error (MS) 0.32 2.89 0.008 0.11 0.002 0.396 0.003 0.155 https://doi.org/10.1371/journal.pone.0266753.t001 Table 2. Yield and yield attributes under different phosphorus levels, farmyard manure, and biofertilizer treatments. No of pod Grain yield (t/ha) Biomass yield (t/ha) Thousand grain weight (g) Grain P content (%) T0-control, 18.6c 3.54d 16.51d 37.34b 0.207a T1-60 kg SSP, 25.9b 4.57ab 19.73ab 39.02a 0.215a T2- farmyard manure (FYM) 2.5 t/ha, 25.9b 4.24bc 18.01c 38.14ab 0.211a T3-2.5 t ha-1 FYM+60 kg SSP 29.7a 4.91a 20.99a 39.0a 0.215a T4-2.5 t ha-1 FYM + 30 kg SSP 26.5b 4.55ab 19.08bc 38.12ab 0.211a T5-consortium biofertilizer 21.5c 3.89cd 17.94c 37.92ab 0.209a T6-consortium biofertilizer +60 kg SSP 28.9ab 4.72a 20.39ab 38.7ab 0.213a T7-consortium biofertilizer +30 kg SSP 25.7b 4.12c 18.32c 37.78ab 0.209a CV 9.57 6.67 5.53 3.10 2.78 Pr>F (treatment) <0.01 <0.01 <0.01 0.21 0.26 Pr>F (Year Treatment) 0.27 0.96 0.32 0.73 0.70 Error (MS) 5.89 0.083 1.09 0.70 0.0003 https://doi.org/10.1371/journal.pone.0266753.t002 Phosphorus and soil physical environment. The accessibility of phosphorus also improves the utilization of N and K in the plant system. As phosphorus is involved in the energy cycle of plant in addition to its involvement in protein and lipids in the plant system, that help in improving plant growth. The data on the root dry matter accumulation in various treatments revealed that the early root growth of black gram at 35 DAS was significantly affected by the different combinations of source of phosphorus, FYM and biofertilizers (Table 2). PLOS ONE The root dry mass at 35 DAS (days after sowing) was higher in the treatments where chemical fertilizer were integrated with the organic and biofertilizers such as T7 and T4 as compared to application biofertilizer, organic manure and chemical fertilizer alone. The root dry matter at the harvesting stage was signifi- cantly higher in the treatment T3 where FYM was applied in addition to the full dose of phos- phorus followed by use of biofertilizer and chemical fertilizers. Choudhary et al., [22] also reported beneficial effects of biofertilizer and organic manure on root growth and develop- ment. The recent studies had showed that phosphorus application enhanced root system resulting in better soil-root contact and finally higher P uptake and absorption of mineral nutrients [23]. Table 1. Effect of phosphorus levels, farmyard manure and biofertilizer on plant height and dry matter accumulation. Treatments Plant height (cm) Root dry matter Leaves dry matter Branches/stem dry matter 30 DAS At harvesting 30 DAS At harvesting 30 DAS At harvesting 30 DAS At harvesting T0-control, 15.8c 26.1d 0.39g 2.94c 1.23g 5.31e 0.84c 4.43g T1-60 kg SSP, 17.3c 30.7abc 0.53d 3.91b 1.46cd 8.31bc 0.91bc 9.54b T2- farmyard Manure (FYM) 2.5 t/ha, 17.7b 29.5bc 0.43f 3.34c 1.60a 7.86c 0.93ab 5.67f T3-2.5 t ha-1 FYM+60 kg SSP 18.8a 32.0a 0.52e 4.85a 1.44de 9.26a 0.93ab 10.04a T4-2.5 t ha-1 FYM + 30 kg SSP 17.4b 30.7abc 0.65b 4.31b 1.53b 8.64abc 1.00a 8.33d T5-consortium biofertilizer 16.4c 28.7c 0.57c 3.89b 1.50bc 6.95d 0.89b 5.31f T6-consortium biofertilizer +60 kg SSP 18.5a 31.3ab 0.37h 4.29b 1.40ef 8.79ab 0.91bc 9.01c T7-consortium biofertilizer +30 kg SSP 17.5b 30.1abc 0.80a 4.03b 1.37f 8.18bc 0.84c 7.22e CV 3.22 5.68 5.57 8.30 3.20 7.96 6.28 5.30 Pr>F (Treatment) <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01 Pr>F (Year Treatment) 0.92 0.24 0.97 0.15 0.08 0.07 0.01 0.19 Error (MS) 0.32 2.89 0.008 0.11 0.002 0.396 0.003 0.155 https://doi.org/10.1371/journal.pone.0266753.t001 Table 2. Yield and yield attributes under different phosphorus levels, farmyard manure, and biofertilizer treatments. 3.1. Effect of different phosphorus levels, FYM and biofertilizer on plant growth and dry matter partition The perusal of the data (Table 1) shows that the plant height was significantly affected by the application of manures, biofertilizers and phosphorus application. The data revealed that max- imum plant height was observed in treatment T3 (18.8 cm), followed by treatment T6 and T4. At harvesting, maximum plant height was recorded in the treatment T3 where 2.5 t/ha FYM was applied along with 60 kg SSP, that was statistically at par with the treatment T4, T6, T7 and T1. The highest plant height under such treatment may be due to improved availability of PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 3 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil PLOS ONE PLOS ONE Phosphorus and soil physical environment. The accessibility of phosphorus also improves the utilization of N and K in the plant system. As phosphorus is involved in the energy cycle of plant in addition to its involvement in protein and lipids in the plant system, that help in improving plant growth. The data on the root dry matter accumulation in various treatments revealed that the early root growth of black gram at 35 DAS was significantly affected by the different combinations of source of phosphorus, FYM and biofertilizers (Table 2). The root dry mass at 35 DAS (days after sowing) was higher in the treatments where chemical fertilizer were integrated with the organic and biofertilizers such as T7 and T4 as compared to application biofertilizer, organic manure and chemical fertilizer alone. The root dry matter at the harvesting stage was signifi- cantly higher in the treatment T3 where FYM was applied in addition to the full dose of phos- phorus followed by use of biofertilizer and chemical fertilizers. Choudhary et al., [22] also reported beneficial effects of biofertilizer and organic manure on root growth and develop- ment. The recent studies had showed that phosphorus application enhanced root system resulting in better soil-root contact and finally higher P uptake and absorption of mineral nutrients [23]. Table 1. Effect of phosphorus levels, farmyard manure and biofertilizer on plant height and dry matter accumulation. PLOS ONE 3.2 Effect of different phosphorus levels, FYM and biofertilizer on yield and yield attributes The data revealed that the number of pods were significantly high (29.7) in the treatment T3 (FYM + 60 kg ha-1 SSP) as compared to T1 and statistically at par with the treatment T6. The thousand-grain weight was also higher in the treatment T1 and T3 that were statistically at par with the treatments T2, T4, T5, T6 and T7. The black gram grain yield was significantly affected by the dose of phosphorus, FYM and biofertilizer application (Table 2). The black gram grain yield was significantly higher in the treatment T3 as compared to control, T2 and T7. The grain yield in T3 was 7.4 and 4.0% higher than the treatment T1 and T6. The application of FYM + 60 kg ha-1 SSP and biofertilizer +60 kg ha-1 SSP has improved the grain yield by 7.4% and 3.28% over the use of 60 SSP alone. The grain yield improvement may be resulted from the improved soil supply of phosphorus with the integrated use of FYM and biofertilizers. Apa´ez Barrios et al., [24] reported that the inade- quate supply of phosphorus affects the photosynthetic processes as well as photosynthates sup- ply to nodules and also adversely affect the root growth, activity and nodule formation. The biomass yield of the black gram was increased with increasing rates of phosphorus application integrated with FYM and biofertilizers up to 60 kg ha-1 SSP. The biomass yield in T3 (20.99) significantly higher than the control and at par with the use of chemical fertilizer alone. The biomass yield with application of FYM + 60 kg SSP (T3) was 6.4 and 2.9% higher than the treatment T1 and T6. Thus, the integrated use of FYM +60 kg ha-1 SSP and biofertili- zer + 60 kg ha-1 SSP has improved the grain yield by 6.4% and 3.34% over the use of 60 SSP (T1) alone. The improvement in biomass yield may be ascribed to the favourable effect of phos- phorus application and other organics on plant height, dry matter production, and number of pods as reported by Niraj and Ved [25] and Parashar et al [26]. PLOS ONE ps //do o g/ 0 3 /jou a po e 0 66 53 g00 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 5 / 10 The leaf dry matter accumulation at 35 DAS was significantly higher in the T2 treatment (1.60 g per plant) followed by T4 (1.50 g per plant) and T3 (1.44 g per plant) as compared to the control and use of chemical fertilizer alone (T1). At harvesting stage, highest leaf dry matter was recorded in the treatmentT3 that was statistically similar to the treatments T4, T6 and T7 and significantly higher than the control and use of 60 kg SSP alone (Fig 1) The stem dry matter at 35 DAS was highest in the treatment T4 (FYM + 30 kg SSP) that was statistically at par with treatment T3 (FYM + 60 kg SSP) and T2 (FYM alone) (Fig 1). The branch dry matter at harvesting stage was highest (10.04 g per plant) in with the application of FYM along with 60 kg SSP (T3), that was statistically superior to use of 60 kg SSP alone (T1). PLOS ONE Yield and yield attributes under different phosphorus levels, farmyard manure, and biofertilizer treatments. No of pod Grain yield (t/ha) Biomass yield (t/ha) Thousand grain weight (g) Grain P content (%) T0-control, 18.6c 3.54d 16.51d 37.34b 0.207a T1-60 kg SSP, 25.9b 4.57ab 19.73ab 39.02a 0.215a T2- farmyard manure (FYM) 2.5 t/ha, 25.9b 4.24bc 18.01c 38.14ab 0.211a T3-2.5 t ha-1 FYM+60 kg SSP 29.7a 4.91a 20.99a 39.0a 0.215a T4-2.5 t ha-1 FYM + 30 kg SSP 26.5b 4.55ab 19.08bc 38.12ab 0.211a T5-consortium biofertilizer 21.5c 3.89cd 17.94c 37.92ab 0.209a T6-consortium biofertilizer +60 kg SSP 28.9ab 4.72a 20.39ab 38.7ab 0.213a T7-consortium biofertilizer +30 kg SSP 25.7b 4.12c 18.32c 37.78ab 0.209a CV 9.57 6.67 5.53 3.10 2.78 Pr>F (treatment) <0.01 <0.01 <0.01 0.21 0.26 Pr>F (Year Treatment) 0.27 0.96 0.32 0.73 0.70 Error (MS) 5.89 0.083 1.09 0.70 0.0003 https://doi.org/10.1371/journal.pone.0266753.t002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 4 / 10 PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil Fig 1. Effect of phosphorus levels, farmyard manure and biofertilizer on biomass accumulation at 35 DAS (a) and At harvesting (b). Bars represents error (MS). https://doi.org/10.1371/journal.pone.0266753.g001 Fig 1. Effect of phosphorus levels, farmyard manure and biofertilizer on biomass accumulation at 35 DAS (a) and At harvesting (b). Bars represents error (MS). https://doi.org/10.1371/journal.pone.0266753.g001 https://doi.org/10.1371/journal.pone.0266753.g001 https://doi.org/10.1371/journal.pone.0266753.g001 The leaf dry matter accumulation at 35 DAS was significantly higher in the T2 treatment (1.60 g per plant) followed by T4 (1.50 g per plant) and T3 (1.44 g per plant) as compared to the control and use of chemical fertilizer alone (T1). At harvesting stage, highest leaf dry matter was recorded in the treatmentT3 that was statistically similar to the treatments T4, T6 and T7 and significantly higher than the control and use of 60 kg SSP alone (Fig 1) The stem dry matter at 35 DAS was highest in the treatment T4 (FYM + 30 kg SSP) that was statistically at par with treatment T3 (FYM + 60 kg SSP) and T2 (FYM alone) (Fig 1). The branch dry matter at harvesting stage was highest (10.04 g per plant) in with the application of FYM along with 60 kg SSP (T3), that was statistically superior to use of 60 kg SSP alone (T1). 3.2 Effect of different phosphorus levels, FYM and biofertilizer on yield and yield attributes oil PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil The grain P content was not significantly affected by the application of phosphorus, FYM and biofertilizers, however grain P content in T3 treatment was 3.86% higher than the control. 3.2 Effect of different phosphorus levels, FYM and biofertilizer on yield and yield attributes The data revealed that the number of pods were significantly high (29.7) in the treatment T3 (FYM + 60 kg ha-1 SSP) as compared to T1 and statistically at par with the treatment T6. The thousand-grain weight was also higher in the treatment T1 and T3 that were statistically at par with the treatments T2, T4, T5, T6 and T7. The black gram grain yield was significantly affected by the dose of phosphorus, FYM and biofertilizer application (Table 2). The black gram grain yield was significantly higher in the treatment T3 as compared to control, T2 and T7. The grain yield in T3 was 7.4 and 4.0% higher than the treatment T1 and T6. The application of FYM + 60 kg ha-1 SSP and biofertilizer +60 kg ha-1 SSP has improved the grain yield by 7.4% and 3.28% over the use of 60 SSP alone. The grain yield improvement may be resulted from the improved soil supply of phosphorus with the integrated use of FYM and biofertilizers. Apa´ez Barrios et al., [24] reported that the inade- quate supply of phosphorus affects the photosynthetic processes as well as photosynthates sup- ply to nodules and also adversely affect the root growth, activity and nodule formation. The biomass yield of the black gram was increased with increasing rates of phosphorus application integrated with FYM and biofertilizers up to 60 kg ha-1 SSP. The biomass yield in T3 (20.99) significantly higher than the control and at par with the use of chemical fertilizer alone. The biomass yield with application of FYM + 60 kg SSP (T3) was 6.4 and 2.9% higher than the treatment T1 and T6. Thus, the integrated use of FYM +60 kg ha-1 SSP and biofertili- zer + 60 kg ha-1 SSP has improved the grain yield by 6.4% and 3.34% over the use of 60 SSP (T1) alone. The improvement in biomass yield may be ascribed to the favourable effect of phos- phorus application and other organics on plant height, dry matter production, and number of pods as reported by Niraj and Ved [25] and Parashar et al [26]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 5 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 . 3.3 Phosphorus availability and uptake under different phosphorus levels, FYM and biofertilizer application The data revealed highest available soil phosphorus in the treatment T3 followed by T4 and T2 (Fig 2B). The soil available phosphorus was highest where FYM was applied along with chemi- cal phosphorus fertilizer. The higher soil available phosphorus in the treatments where FYM was applied may be due to addition of extra phosphorus through the FYM and decomposition of FYM produces organic acids which might had improved the availability of phosphorus in the soil. The biofertilizers has also improved the P availability due to phosphorus solubilizing action of bacteria’s and fungi applied to the seeds. The soil availability has affected the phosphorus uptake of black grams (Fig 2A). A signifi- cantly higher P uptake was observed in the treatment T3 (26.4 kg/ha) as compared to control. The phosphorus uptake in treatment with application of FYM along with 60 kg ha-1 SSP (T3) was statistically at par with treatment T6 and T1. The improved soil availability may be resulted from the application of fertilizer, whereas application of fertilizer along with FYM and bioferti- lizer was more efficient in raising the soil available phosphorus. The microbes from the biofer- tilizer and organic acids from the FYM decomposition converts unavailable phosphorus to available form. 4. Discussion Phosphorus plays important role in metabolic processes associated with shooting organs, energy generation, synthesis of nucleic acid, photosynthesis, and respiration of legume crops [27–29]. The P have crucial role in nodule energetic transformations thus legumes require more phosphorus as compared to cereals [30]. Phosphorus is found in soil as primary P miner- als (apatite) and secondary clay minerals, such as calcium, iron, and aluminium phosphates, which are important in maintaining phosphorus buildup and availability through desorption and dissolution processes [31]. With the application of farmyard manure and biofertilizer, the field experiment revealed an increase in plant height, number of pods, root and shoot biomass, and root and shoot biomass (Tables 1 and 2). The integrated application of organics and fertil- izers has increased the thousand grain weight by enhancing flowering and seed formation [32, 33]. The farmyard manure releases phosphorus and other macro and micronutrients upon Fig 2. (a) Phosphorus uptake under different treatments (Bars represents standard error) (LSD = 1.92) 2 (b) Soil available phosphorus under different treatments before harvesting. https://doi.org/10.1371/journal.pone.0266753.g002 Phosphorus plays important role in metabolic processes associated with shooting organs, energy generation, synthesis of nucleic acid, photosynthesis, and respiration of legume crops [27–29]. The P have crucial role in nodule energetic transformations thus legumes require more phosphorus as compared to cereals [30]. Phosphorus is found in soil as primary P miner- als (apatite) and secondary clay minerals, such as calcium, iron, and aluminium phosphates, which are important in maintaining phosphorus buildup and availability through desorption and dissolution processes [31]. With the application of farmyard manure and biofertilizer, the field experiment revealed an increase in plant height, number of pods, root and shoot biomass, and root and shoot biomass (Tables 1 and 2). The integrated application of organics and fertil- izers has increased the thousand grain weight by enhancing flowering and seed formation [32, 33]. The farmyard manure releases phosphorus and other macro and micronutrients upon mpared t ary clay maintain es [31]. W d an inc Fig 2. (a) Phosphorus uptake under different treatments (Bars represents standard error) (LSD = 1.92) 2 (b) Soil available phosphorus under different treatments before harvesting. 5. Conclusions The results revealed significant improvement in yield, soil phosphorus availability and phos- phorus uptake in black gram. The integrated use of organic sources such as farm yard manure and consortium biofertilizers improved the phosphorus availability and uptake. The use of farm yard manure and biofertilizer in combination with 60 kg ha-1 SSP increased grain output by 7.4% and 3.28%, respectively, above the use of 60 kg ha-1 SSP alone. Thus, combined use of organic and inorganic chemical fertilizers may be recommended instead of chemical fertilizer alone to reduce the dependence on chemical fertilizers, to have improvement in soil health for sustainable production of black grams in the region. Acknowledgments The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP-2021/186), King Saud University, Riyadh, Saudi Arabia. Authors thank the Punjab Agricultural University Regional Research Station, Gurdaspur for providing field and laboratory facilities for conducting the experiment. 4. Discussion PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 6 / 10 PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil decomposition, in addition it dissolves the unavailable form of phosphorus through the release of organic acids on decomposition of farmyard manures [31]. The improved availability of phosphorus in soil has been observed (Fig 2B) in response to application of farmyard manure and biofertilizer. The magnitude of availability was higher in the plots with addition of farm- yard manures as compared to initial phosphorus status in the soil. The use of biofertilizers has also improved the phosphorus availability to plants due to the mineralization, solubilization and translocation action of phosphorus solubilizing through production of organic acid and proton extrusion [34, 35]. Nahas [36] attributed increase in solubilization of insoluble phos- phate due to organic acids produced by microbes to the drop in soil pH, soil cations chelating, and competition with phosphate for adsorption sites in the soil solution. The increased avail- ability of phosphorus in soil has also resulted in the improvement in phosphorus uptake in black gram (Table 2). Ganesan [37] also reported improved root and shoot growth of plant along with increased P uptake in black gram compared to untreated plants. Kaur and Reddy [38] and Shahzad et al. [39] also observed improved soil P status and plant P nutrition with inoculation with phosphate solubilizing bacteria resulted from higher alkaline phosphatase activity. Khatkar et al [40] also observed yield improvement with microbial inclulation of urd- bean seeds. Thus, addition of farmyard manure not only add up to nutrient pools but also cre- ates favourable root zone environment for better root activity and nutrient uptake. Likewise, inoculation of black gram seed with consortium biofertilizer improves soil phosphorus avail- ability and plant uptake. Mahanta et al [41] reported inoculation of soyabean seed with phos- phorus solublizing bacteria and VAM may substitute about 50% phosphatic fertilizer under soyabean-wheat crop rotation, similar results have been found with black gram in the present study. References 1. Gour YD, Microbiology, physiology and agronomy of nitrogen fixation: Legume-Rhizobium symbiosis. Proc. Indian Nat Sci Acad, 1993; 59:333–358. 2. Anonymous 2018, Agricultural statistics at a glance 2018. Directorate of Economics and Statistics, Department of Agriculture and Cooperation, Govt. of India; 2018 New Delhi. 3. Bargaz A, Faghire M, Abdi N, Farissi M, Sifi B, Drevon JJ, et al. Low soil phosphorus availability increases acid phosphatases activities and affects P partitioning in nodules, Seeds and Rhizosphere of Phaseolus vulgaris. Agriculture 2012; 2:139–153. https://doi.org/10.3390/agriculture2020139 3. Bargaz A, Faghire M, Abdi N, Farissi M, Sifi B, Drevon JJ, et al. Low soil phosphorus availability increases acid phosphatases activities and affects P partitioning in nodules, Seeds and Rhizosphere of Phaseolus vulgaris. Agriculture 2012; 2:139–153. https://doi.org/10.3390/agriculture2020139 4. Zhu F, Qu L, Hong X, Sun X, Isolation and characterization of a phosphate-solubilizing halophilic bacte- rium Kushneria sp. YCWA18 from Daqiao Saltern on the coast of yellow sea of China. Evid Based Com- plement Alternat Med, 2011, 6. https://doi.org/10.1155/2011/615032 PMID: 21716683 4. Zhu F, Qu L, Hong X, Sun X, Isolation and characterization of a phosphate-solubilizing halophilic bacte- rium Kushneria sp. YCWA18 from Daqiao Saltern on the coast of yellow sea of China. Evid Based Com- plement Alternat Med, 2011, 6. https://doi.org/10.1155/2011/615032 PMID: 21716683 5. Sepetoglu H, Grain legumes. Department of Field Crops, Faculty of Agriculture, University of Ege, Yzmir, Turkey. 2002. 5. Sepetoglu H, Grain legumes. Department of Field Crops, Faculty of Agriculture, University of Ege, Yzmir, Turkey. 2002. 6. Marschner P, Marschner’s Mineral Nutrition of Higher Plants. 3rd Edition, Academic Press, San Diego, 2012; p: 651. 7. Yugandhar P, Savithramma N, Green synthesis of calcium carbonate Nanoparticles and their effects on seed germination and seedling growth of Vigna Mungo. Int J Advanced Res, 2013; 1(8): 89–103. 8. Tessema T, Alemayehu B, Effect of Phosphorus Application and Varieties on grain yield and yield com- ponents of Common Bean (Phaseolus vulgaris L.). Am J Plant Nutr Ferti Tech, 2015; 5:79–84. 9. Devi KN, Singh LNK, Devi TS, Devi HN, Singh TB, Singh KK, et al. Response of soybean [Glycine max (L.) Merrill] to sources and levels of phosphorus. J Agri Sci 2012; 4: 44–53. 10. 10. Tekle E, Kubure, Raghavaiah CV, Ibrahim H, Production Potential of Faba Bean (Vicia faba L.) Geno- types in Relation to Plant Densities and Phosphorus Nutrition on Vertisols of Central Highlands of West Showa Zone, Ethiopia, East Africa. Author Contributions Conceptualization: Jagdish Singh, Rajan Bhatt, B. S. Dhillon. Data curation: B. S. Dhillon, Asma A. Al-Huqail. Formal analysis: Jagdish Singh, Dhaliwal S. S., B. S. Dhillon, Asma A. Al-Huqail, Alanoud Alfagham, Manzer H. Siddiqui, Hayssam M. Ali, Faheema Khan, Ritesh Kumar. PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 7 / 10 Funding acquisition: Dhaliwal S. S., B. S. Dhillon, Asma A. Al-Huqail, Alanoud Alfagham, Manzer H. Siddiqui, Faheema Khan. Investigation: Rajan Bhatt. Methodology: Jagdish Singh, B. S. Dhillon. Project administration: Jagdish Singh, B. S. Dhillon. Resources: Jagdish Singh, Rajan Bhatt, Dhaliwal S. S., Alanoud Alfagham, Hayssam M. Ali, Faheema Khan. Software: Jagdish Singh, Rajan Bhatt, Asma A. Al-Huqail, Manzer H. Siddiqui, Hayssam M. Ali, Ritesh Kumar. Validation: Jagdish Singh, Rajan Bhatt. Visualization: Jagdish Singh, Rajan Bhatt, Dhaliwal S. S., B. S. Dhillon. Writing – original draft: Jagdish Singh, Rajan Bhatt, Dhaliwal S. S., B. S. Dhillon, Asma A. Al- Huqail. Writing – review & editing: Rajan Bhatt, Asma A. Al-Huqail, Alanoud Alfagham, Hayssam M. Ali, Faheema Khan, Ritesh Kumar. R f Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil Methodology: Jagdish Singh, B. S. Dhillon. Project administration: Jagdish Singh, B. S. Dhillon. Resources: Jagdish Singh, Rajan Bhatt, Dhaliwal S. S., Alanoud Alfagham, Hayssam M. Ali, Faheema Khan. Software: Jagdish Singh, Rajan Bhatt, Asma A. Al-Huqail, Manzer H. Siddiqui, Hayssam M. Ali, Ritesh Kumar. Validation: Jagdish Singh, Rajan Bhatt. Visualization: Jagdish Singh, Rajan Bhatt, Dhaliwal S. S., B. S. Dhillon. Writing – original draft: Jagdish Singh, Rajan Bhatt, Dhaliwal S. S., B. S. Dhillon, Asma A. Al- Huqail. Writing – review & editing: Rajan Bhatt, Asma A. Al-Huqail, Alanoud Alfagham, Hayssam M. Ali, Faheema Khan, Ritesh Kumar. 14. Soumare M, Tack FMG, Verloo MG, Effects of a municipal solid waste compost and mineral fertilization on plant growth in two tropical agricultural soils of Mali. Bioresour. Tech. 2003; 86:15–20. https://doi. org/10.1016/s0960-8524(02)00133-5 PMID: 12421002 References Advances Crop Sci Tech 2016; 4:214. 11. Ullah S, Jan A, Ali M, Ahmad A, Ullah A, Ahmad G, et al. Effect of phosphorous under different applica- tion methods on yield attributes of chickpea (cicer arietinum L.). Int J Agri Environ Res 2017; 3: 79–85. 12. Karikari B, Arkorful E, Addy S, Growth, Nodulation and Yield Response of Cowpea to phosphorus fertil- izer application in Ghana. J Agron 2015; 14: 234–240. 13. Nambiar KKM, Abrol IP, Long term fertilizer experiments in India-An over view. Fertilizer News. 1992; 34: 11–20. 14. Soumare M, Tack FMG, Verloo MG, Effects of a municipal solid waste compost and mineral fertilization on plant growth in two tropical agricultural soils of Mali. Bioresour. Tech. 2003; 86:15–20. https://doi. org/10.1016/s0960-8524(02)00133-5 PMID: 12421002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 8 / 10 PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil 15. Mo¨ller K, Influence of different manuring systems with and without biogas digestion on soil organic mat- ter and nitrogen inputs, flows and budgets in organic cropping systems. Nutr Cycl Agroecosyst 2009; 84:179–202. 16. Swarup A, Lessons from long term fertilizer experiments in improving fertilizer use efficiency and crop yields. Fert News 2002; 47(12):59–73. 17. Syers JK, Johnston AE, Curtin D (2008) Efficiency of soil and fertilizer phosphorus use—reconciling changing concepts of soil phosphorus behaviour with agronomic information, FAO Fertilizer and Plant Nutrition Bulletin 18. FAO, United Nations, Rome. 18. Roberts TL, Johnston AE, Phosphorus use efficiency and management in agriculture. Resour Conser Recycl 2015; 105:275–281. 19. Ahemad M, Kibret M. Mechanisms and applications of plant growth promoting rhizobacteria: current perspective. J King Saud Univ Sci 2014; 26(1):1–20. 20. Shree S, Singh VK, Ravi K, Effect of integrated nutrient management on yield and quality of cauliflower. The Bioscan 2014, 9(3):1053–1058. 21. Chapman H D, and Pratt P F, Methods of analysis for soil plant and waters. Berkley: University of Cali- fornia, Division of Agriculture. 1961. pp 150–179. 22. Choudhary R, Singh K, Manohar RS, Yadav AK, Sangwan A. Response of different sources and levels of phosphorus on yield, nutrient like uptake and net returns on mung bean under rain fed condition. Indian J Agric Res, 2015; 35: 263–268. 23. Zafar M, Abbasi M, Rahim N, Khaliq A, Shaheen A, Jamil M, et al. References Influence of integrated phosphorus supply and plant growth promoting Rhizobacteria on growth, nodulation, yield and nutrient uptake in Phaseolus vulgaris. Afr J Biotechnol 2011; 10(74):16793–16807. 24. Apa´ez Barrios P, Escalante Estrada JA, Gonza´lez R, Cha´vez M, Analysis of Cowpea Growth and Pro- duction in Maize Trellis with Nitrogen and Phosphorus. Int J Agri Sci, 2014; 4: 102–108. 25. Niraj VPS, Ved P, Effect of phosphorus and sulphur on growth, Yield and quality of black gram (Phaseo- lus mungo.), Asian J Soil Sci, 2014, 9(1):117–120. 26. Parashar A, Jain M, Tripathi L, Effect of Sulphur and Phosphorus on the Growth and Yield of Black Gram (Vigna mungo L.), Ind J Pure App Biosci, 2020; 8(5): 276–280. 27. Vance C. P, Uhde-Stone C, Allan D. L, Phosphorus acquisition and use: critical adaptations by plants for securing a nonrenewable resource. New Phytol 2003; 157: 423–447. https://doi.org/10.1046/j.1469- 8137.2003.00695.x PMID: 33873400 28. Chaudhary MI, Adu-Gyamfi JJ, Saneoka H, Nguyen NT, Suwa R, Kanai S, et al. Lightfoot DA, Fujita K, Effect of phosphorus deficiency on nutrient uptake nitrogen fixation and photosynthetic rate in mash- bean, mungbean and soybean. Acta Physiol Plant 2008; 30: 537–544. 29. Zhang Z, Liao H, Lucas WJ, Molecular mechanisms underlying phosphate sensing, signaling, and adaptation in plants. J Integr Plant Biol 2014; 56: 192–220. https://doi.org/10.1111/jipb.12163 PMID: 24417933 30. Rotaru V, Sinclair TR, Interactive influence of phosphorus and iron on nitrogen fixation by soybean. Environ Exp Bot 2009; 66(1): 94–99. 31. Mitran T, Mani PK, Effect of organic amendments on rice yield trend, phosphorus use efficiency, uptake, and apparent balance in soil under long-term rice-wheat rotation. J Plant Nutr, 2017; 40(9):1312–1322. 32. Ullah A, Ali A, Waseem M, Nadeem MA, Tahir M, Iqbal A, et al. Response of two mung bean cultivars with different phosphorus levels under Faisalabad condition. Int J Applies Agric Res, 2010; 5(5):621– 628. 33. Verma G, Kumawat N, Morya J, Nutrient management in mung bean [Vigna radiate (L.) Wilczek] for higher production and productivity under semi-arid tract of central India. Int J Curr Microbial App Sci, 2014; 6(7):488–493. 34. Marra LM, de Oliveira SM, Soares CRFS, de Souza Moreira FM, Solubilisation of inorganic phosphates by inoculants strains from tropical legumes. Scientia Agricola, 2011, 68:603–609. 35. Gupta G, Panwar J, Jha P, Natural occurrence of Pseudomonas aeruginosa, a dominant cultivable Dia- zotrophic endophytic bacterium colonizing Pennisetum glaucum (L) R. Br. Appl Soil Ecol, 2013; 64:252–261. 36. References Nahas E, Factors determining rock phosphate solubilization by microorganism isolated from soil. World J Microbiol Biotechnol, 1996; 12:18–23. 37. Ganesan V, Rhizoremediation of cadmium soil using a cadmium-resistant plant growth promoting rhi- zopseudomonad. Current Microbiol, 2008; 56:403–407. https://doi.org/10.1007/s00284-008-9099-7 PMID: 18239844 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 9 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 38. Kaur G, Reddy MS, Influence of P-solubilizing bacteria on crop yield and soil fertility at multi locational sites. European J Soil Biol, 2014; 61:35–40. 39. Shahzad SM, Khalid A, Arif MS, Riaz M, Ashraf M, Iqbal Z, Co-inoculation integrated with P-enriched compost improved nodulation and growth of chickpea (Cicer arietinum L.) under irrigated and rainfed farming systems. Biol Fertil Soils, 2014; 50:1–12. 40. Khatkar R, Abraham T, Joseph AS, Effect of Bio fertilizers and sulphur levels on growth and yield of black gram (Vigna mungo L.). Legume Res, 2007; 30(3):233–234. 41. Mahanta D, Rai R K, Mishra S D, Raja A, Purakayastha T J and Varghese E Influence of phosphorus and biofertilizers on soybean and wheat root growth and properties. Field Crop Res 2014. 166: 1–9. Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil PLOS ONE Integrated use of fertilizers improves the yield and phosphorus uptake of black gram in silt loam soil 38. Kaur G, Reddy MS, Influence of P-solubilizing bacteria on crop yield and soil fertility at multi locational sites. European J Soil Biol, 2014; 61:35–40. 39. Shahzad SM, Khalid A, Arif MS, Riaz M, Ashraf M, Iqbal Z, Co-inoculation integrated with P-enriched compost improved nodulation and growth of chickpea (Cicer arietinum L.) under irrigated and rainfed farming systems. Biol Fertil Soils, 2014; 50:1–12. 40. Khatkar R, Abraham T, Joseph AS, Effect of Bio fertilizers and sulphur levels on growth and yield of black gram (Vigna mungo L.). Legume Res, 2007; 30(3):233–234. 41. Mahanta D, Rai R K, Mishra S D, Raja A, Purakayastha T J and Varghese E Influence of phosphorus and biofertilizers on soybean and wheat root growth and properties. Field Crop Res 2014. 166: 1–9. PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 10 / 10 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 PLOS ONE \| https://doi.org/10.1371/journal.pone.0266753 April 14, 2022 10 / 10
https://openalex.org/W4282833792	https://zenodo.org/records/6641011/files/Rusu_Viorel%20(1).%20REGIMUL%20DE%20INTEGRITATE%20%C3%8EN%20SERVICIUL%20PUBLIC%20%C3%8EN%20CONDI%C8%9AIILE%20NOII%20LEGISLA%C8%9AII.%20REALIT%C4%82%C8%9AI%20%C8%98I%20PROVOC%C4%82RI.pdf	Romanian, Moldavan	null	REGIMUL DE INTEGRITATE ÎN SERVICIUL PUBLIC ÎN CONDIȚIILE NOII LEGISLAȚII. REALITĂȚI ȘI PROVOCĂRI	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	3,089	Viorel RUSU Lector universitar, Universitatea de Stat din Moldova Lector universitar, Universitatea de Stat din Moldova CZU: 340.134:330.564.22:352/354.08 REGIMUL DE INTEGRITATE ÎN SERVICIUL PUBLIC ÎN CONDIȚIILE NOII LEGISLAȚII. REALITĂȚI ȘI PROVOCĂRI INTEGRITY REGIME IN THE PUBLIC SERVICE UNDER THE NEW LEGISLATION. REALITIES AND CHALLENGES. Viorel RUSU Lector universitar, Universitatea de Stat din Moldova Viorel RUSU Lector universitar, Universitatea de Stat din Moldova Summary 2] nu sunt coerente, previzibile și pun povară suplimentară considerabilă anume pe subiecții declarării lipsiți de asistență și capacități, cum ar fi aceiași funcționarii publici și demnitarii la nivelul local; 2) noile concepte privind „stilul de viață„ sau servicii procurate ce depășesc 10 salarii medii lunare pe economie pe parcursul anului precum și „diferența substanțială” care depășește 20 salarii medii lunare pe perioada cât persoana este subiect al declarării [4, art. 2] nu sunt coerente, previzibile și pun povară suplimentară considerabilă anume pe subiecții declarării lipsiți de asistență și capacități, cum ar fi aceiași funcționarii publici și demnitarii la nivelul local; p ți ș ț p ș 3) mecanismul de suspendare din funcție [5] este la fel unul excesiv și poate crea probleme în funcționarea sistemică a serviciului public; p ți ș ț p ș 3) mecanismul de suspendare din funcție [5] este la fel unul excesiv și poate crea probleme în funcționarea sistemică a serviciului public; 4) sistemul de control, constatare și răspundere în domeniul integrității este în mare parte deconectat de la sistemul existent de drept în domeniul răspunderii juridice. Aceasta creează incertitudine în aplicarea răspunderii juridice pentru în încălcările din domeniul integrității, continuând să persiste riscul ca pentru fapte formale și neesențiale din punct de vedere a interesului public se aplică aceiași sancțiune (pedeapsă) de încetarea a mandatului/funcției și privarea de a ocupa funcții publice pe o anumită perioadă, contrară principiului individualizării răs punderii. p 5) se impun mecanisme de contracarare a abuzurilor de denunțuri și petiții în scopuri politice în legătură cu perioada electorală, etc., luarea în considerație a statutului de persoană aleasă pentru un anumit mandat (cum ar fi de exemplu ales local) la aplicarea măsurilor de răspundere și a altor măsuri de constrângere. Cu regret aceste aspecte nu au fost luate în considerație în noua legislație. 5) se impun mecanisme de contracarare a abuzurilor de denunțuri și petiții în scopuri politice în legătură cu perioada electorală, etc., luarea în considerație a statutului de persoană aleasă pentru un anumit mandat (cum ar fi de exemplu ales local) la aplicarea măsurilor de răspundere și a altor măsuri de constrângere. Cu regret aceste aspecte nu au fost luate în considerație în noua legislație. Astfel constatăm că asigurarea principiului proporționalității dintre intere sul public în domeniu integrității și garantarea dreptului de a fi ales și de a ocu pa o funcției publică lasă de dorit. Legea nr. Summary Public service and integrity regime in public sector are inextricably linked elements. Between the integrity regime and the status of public servants and dignitaries there must be proportionality (balance) regarding the protection of the public interest and the guaran tee of the right to be elected or to hold a public function. In order to guarantee the material and procedural rights of public servants and dignitaries, a clear link is required between the legislation in the field of integrity and the legislation governing legal liability, which is mostly codified and includes disciplinary, contravention and criminal liability, as well as patrimonial liability in connection with mentioned forms. It is interesting to what extent new changes in integrity legislation meet these standards or rigors. Keywords: integrity regime, public service, public servant, dignitary, public inte rest, legal liability. Prin Legea nr. 130/2021 pentru modificarea unor acte normative [1] a fost mo dificată legislația în domeniul integrității, care a intrat în vigoare la 29 octombrie 2021. Noua legislația efectiv a înăsprit rigorile și așa incerte privind declararea ave rii și intereselor personale, regimul conflictelor de interese, al incompatibilităților, restricțiilor și limitărilor (notă: ceea ca în ansamblu lor ar constitui în ceea mai mare parte domeniul integrității) pentru agenții publici, partea covârșitoare con stituind-o funcționarii publici și persoanele ce exercită o funcție de demnitate pu blică (demnitari) [2, art. 3]. În ansamblu situația în domeniul integrității din ultima perioadă este de așa natură că marea majoritate a cazurilor examinate de Autoritatea Națională de Integritate (ANI) privesc aleșii și funcționarii locali (peste 50 %) [3, p.12-16], deși la nivel central sunt concentrate resursele financiare și există cauze de rezonanță majoră care nu s-au soldat cu finalitate. Analizând procesul de elaborare, consultare și adoptare a pachetului de legi din domeniul integrității citat mai sus, pot fi evidențiate următoarele deficiențe: 1) graba și neconsultarea adecvată a tuturor actorilor, lipsa analizei per ansamblu (lecțiile învățate), inclusiv costul sistemului existent. Aceasta ar pu tea agrava și mai mult neatractivitatea activității în serviciul public, în special la 379 nivel local care deja se confruntă cu majore probleme de personal (inclusiv ca o consecință a depopulării țării); 2) noile concepte privind „stilul de viață„ sau servicii procurate ce depășesc 10 salarii medii lunare pe economie pe parcursul anului precum și „diferența substanțială” care depășește 20 salarii medii lunare pe perioada cât persoana este subiect al declarării [4, art. Summary 132/2016 cu privire la Autoritatea Națională de Integritate prevede că inspectorul de integritate decide dacă emite un act de constatare în care solicită încetarea mandatului, a raporturilor de muncă sau de serviciu ale subiectului declarării și aplică pentru acesta interdicția de a ocupa o funcție publică sau de demnitate publică în funcție de gradul de vinovăție al subiectului declarării, de natura abaterii, de numărul și durata abaterilor, de comiterea unor abateri similare anterior, de prejudiciul adus interesului public și de funcția publică deținută. Iar Autoritatea Națională de Integritate se adresea ză instanței de judecată pentru declararea nulității actului administrativ, a actu lui juridic sau a deciziei luate cu încălcarea dispozițiilor legale privind conflicte le de interese, incompatibilitățile restricțiile și limitările, dacă anularea acestora nu aduce daune interesului public. La aprecierea faptului dacă sunt aduse daune interesului public se va ține cont în principal de caracterul sistematic sau unitar al abaterii, de numărul de persoane afectate, de mărimea prejudiciului material cauzat, de consecințele abaterii la nivel local, național sau internațional, de afec tarea reputației autorității/instituției publice sau a bunei funcționări a acesteia, precum și de alte circumstanțe care ar afecta interesul public[6]. Respectiv Legea 380 nr. 133/2016 privind declararea averii și a intereselor personale prevede că actele administrative emise/adoptate sau actele juridice încheiate personal sau prin in termediul unei persoane terțe într-o situaţie de conflict de interese real sunt lovite de nulitate absolută, cu excepţia cazului în care anularea acestora ar aduce daune interesului public[7]. Cele menționate atestă că legislația în domeniul integrității nu este încadrată în sistemul de drept existent privind răspunderea juridică. p p p j Legislația ce reglementează răspunderea juridică în cea mai mare parte este codificată și include răspunderea disciplinară, contravențională și penală, precum și patrimonială în conexiune cu formele menționate. Această legislație este comple xă în aspectul normelor materiale și procedurale, inclusiv garantarea drepturilor procesuale ale persoanei. Lucrul respectiv nu-l putem spune despre noua legislație în domeniul integrității (notă: cu atât mai mult despre legislația precedentă), care rămâne a fi imprevizibilă în aspectul termenilor, procedurilor, individualizării răspunderii (circumstanțe atenuante, agravante, statutul funcționarului (dem nitarului) etc.) și asigurării garanțiilor procesuale necesare conform principiului dreptății. Summary De exemplu conform căror norme și în ce condiții (standarde) inspecto rul de integritate (ANI) va garanta emiterea „unui act de constatare în care solicită încetarea mandatului, a raporturilor de muncă sau de serviciu ale subiectului de clarării și aplică pentru acesta interdicția de a ocupa o funcție publică sau de dem nitate publică în funcție de gradul de vinovăție al subiectului declarării, de natu ra abaterii, de numărul și durata abaterilor, de comiterea unor abateri similare anterior, de prejudiciul adus interesului public și de funcția publică deținută”? De asemenea care sunt garanțiile că se va determina adecvat că sunt „aduse daune interesului public și se va ține cont în principal de caracterul sistematic sau unitar al abaterii, de numărul de persoane afectate, de mărimea prejudiciului material ca uzat, de consecințele abaterii la nivel local, național sau internațional, de afectarea reputației autorității/instituției publice sau a bunei funcționări a acesteia, precum și de alte circumstanțe care ar afecta interesul public”? Respectiv mecanisme legale lipsesc și rămâne la latitudinea pur subiectivă a reprezentatului ANI de a decide într-un mod sau altul. La rândul său procedura ulterioară în instanța de judecată în procedura de contencios administrativ nu este adaptată și are alte sarcini decât individualizarea și aplicarea conform principiului dreptății a răspunderii juridice sub formă de „încetare a mandatului, a raporturilor de muncă sau de serviciu ale subiectului declarării și interdicția de a ocupa o funcție publică sau de demnitate publică”. Efectiv acestea sunt sancțiuni contravenționale sau penale iar „raporturi le juridice ale autorităților publice care acționează în baza Codului contravențional sau Codului penal nu cad sub incidența Codului administrativ”, adică în speță pro cedura contenciosului administrativ[8]. Pre ederile legislației în domeniul integrității ar trebui să țină cont de statu Prevederile legislației în domeniul integrității ar trebui să țină cont de statu tul persoanelor care sunt alese prin mandat direct de către cetățeni (cum ar fi de exemplu aleșii locali). Astfel considerăm că un demnitar poate fi lipsit de mandatul acordat de cetățean doar în cazuri de excepții pentru fapte ce constituie infracțiuni, 381 în restul situațiilor existând sancțiuni contravenționale de privare pe un anumit termen, etc.. Summary Prezintă interes și chiar câmp de discuție, inclusiv în contextul unor situații care au avut loc (notă: actul de constatare ANI în privința Președintelui Curții Constituționale[9]), dacă mandatul Președintelui Republicii Moldova, care de asemenea este un demnitar ales, poate înceta prin actul de constatare a funcționar cu statutul special (inspectorul de integritate) dintr-o autoritate admi nistrativă autonomă (ANI). Respectiv cum se încadrează aceasta cu prevederile constituționale de încetarea a mandatului Președintelui Republicii Moldova? Re spectiv funcțiile de demnitate publică obținute prin mandat direct, în unele cazuri și indirect, nu pot fi echivalate cu funcțiile publice ordinare ori executive. În contextul celor menționate, nu suferă nici o critică prevederile art. 23 din Legea privind declararea averii şi a intereselor personale[10]. Conform ali neatului (1) din articolul respectiv „încălcarea prevederilor prezentei legi atrage răspundere disciplinară, contravențională, civilă sau penală, după caz”, iar ali neatul (3) din același articol prevede că „fapta subiectului declarării în privința căruia s-a constatat starea de incompatibilitate nesoluționată în termenul legal constituie temei pentru tragere la răspundere disciplinară, contravențională și/ sau, după caz, pentru încetarea mandatului, a raporturilor de muncă ori de ser viciu ale acestuia.”. Rămâne neclară la care din formele menționate de răspun dere juridică (de la alineatul (1)) se atribuie „și/sau, după caz, pentru încetarea mandatului, a raporturilor de muncă ori de serviciu ale acestuia”? Alte exemple de incertitudini similare pot fi continuate și în cazul altor prevederi din articolul și legislația din domeniul integrității cum ar fi: ”subiectul declarării eliberat sau destituit din funcţie potrivit prevederilor alin. (3)–(5) este decăzut din dreptul de a mai exercita o funcţie publică sau o funcţie de demnitate publică, cu excepţia funcţiilor eligibile, pe o perioadă de 3 ani de la data eliberării sau destituirii din funcţia publică ori de demnitate publică respectivă sau din data încetării de drept a mandatului său. Dacă persoana a ocupat o funcţie eligibilă, ea nu mai poate ocu pa aceeaşi funcţie pe o perioadă de 3 ani de la încetarea mandatului.” Respectiv legislația din domeniul integrității admite aplicarea a 2 sau chiar 3 sancțiuni pen tru aceiași faptă (!?). Aceste prevederi încalcă principiile dreptății, previzibilității, individualizării răspunderii și ordinii aplicării normelor de drept. Astfel actul ANI de constatare se transformă în act de sancționare, deși sancționarea ar tre bui efectuată în limitele unei proceduri disciplinare contravenționale sau penale bine reglementate. Summary La fel, în contextul celor menționate, suspendarea din funcție ar trebui să aibă loc în cadrul unor proceduri disciplinare, contravenționale sau penale cu temeiuri foarte clare privind necesitatea acestor măsuri de constrângere. Considerăm că Actul de constatare al Autorității Naționale de Integritate tre buie să fie un act de control[11], iar în baza acestuia (reieșind din caracterul faptei), inspectorul de integritate sau inițiază o procedură de răspundere disciplinară adre sată autorității competente de angajare, ori întocmește un proces-verbal cu privire 382 la contravenție (notă: componențele de contravenții trebuie adaptate reieșind din caracterul faptei și al subiectului declarării), sau transmite materialele organului de urmărire penală pentru atragerea la răspundere penală. Atribuindu-se la una din categoriile de procedură administrativă activita tea administrativă și actul de constatare ANI trebuie să aibă termene foarte bine definite[12]. Se impune respectarea unor așa principii ale procedurii administra tive ca eficiența, termenul rezonabil, proporționalitatea, securitatea raporturilor juridice[13]. Cât privește averea nejustificată legislația contravențională și cea penală are deja ca măsură de siguranță confiscarea specială și această măsură de constrângere ar trebui încadrată armonios în legislația respectivă[14], care corespunde elemen tului superior al sistematizării unitare a cadrului normativ - codificarea. Sistemul instanțelor judecătorești ce examinează cauzele în domeniul integrității (în principal actele de constatare al ANI) nu respectă principiul subsidiarității ca un principiu cheie în cadrul Uniunii Europene[15]. Astfel actele de constatare ale ANI pot fi contestate la Curtea de Apel Chișinău, în termen de 30 de zile de la emitere[16]. Dacă admite recursul împotriva hotărârii Curții de Apel Chișinău, Curtea Supremă de Justiție nu poate trimite cauza la rejudecare[17]. Este evident câte cheltuieli și resurse trebuie să suporte funcționarul sau demnitarul public care domiciliază în afara razei orașului Chișinău pentru a-și apăra drepturile în instanță pe cazurile din domeniul integrității. Finalmente considerăm că se impune reevaluarea și revizuirea prevederilor legislației în domeniul integrității având la bază balanța dintre drepturile și intere sele persoanei care activează în serviciul public, pe de o parte, și concentrarea pe criteriile de risc, adică pe mecanisme eficiente pentru persoane și funcții publice cu impact major, pe de altă parte. Cu regret legislația în domeniul integrității continuă practica de „împovărare” pentru majoritatea funcționarilor și demnitarilor public, altfel spus există riscul de-motivării de a activa în serviciul și sectorul public. Referințe bibliografice și resurse Web: A se vedea Art. I și II din Legea pentru modificarea unor acte normati 1. ve, nr. 130 din 07 octombrie 2021 [citat 06.12.2021]. Disponibil: https:// www.legis.md/cautare/getResults?doc_id=128365&lang=ro. A se vedea Art. I și II din Legea pentru modificarea unor acte normati 1. ve, nr. 130 din 07 octombrie 2021 [citat 06.12.2021]. Disponibil: https:// www.legis.md/cautare/getResults?doc_id=128365&lang=ro. g g g Legea integrității, nr. 82 din 25 mai 2017 [citat 07.12.2021] Disponibil: 2. https://www.legis.md/cautare/getResults?doc_id=120706&lang=ro. Legea integrității, nr. 82 din 25 mai 2017 [citat 07.12.2021] Disponibil: 2. https://www.legis.md/cautare/getResults?doc_id=120706&lang=ro. Raportul de activitate al Autorității Naționale de Integritate pentru anul 3. 2021 [citat 07.12.2021] Disponibil: https://ani.md/sites/default/files/Ra port%20ANI%202021.pdf. Legea privind declararea averii și a intereselor personale, nr. 133 din 17 4. iunie 2016 [citat 05.12.2021] Disponibil: https://www.legis.md/cautare/ getResults?doc_id=128397&lang=ro. 383 A se vedea art. 7 alin. (2) lit. c) din Legea cu privire la Autoritatea Națională 5. de Integritate, nr. 132 din 17 iunie 2016 [citat 07.12.2021] Disponibil: https://www.legis.md/cautare/getResults?doc_id=128394&lang=ro. A se vedea art. 39 alin. (2), (4) lit. b) și (5) din Legea cu privire la Au 6. toritatea Națională de Integritate, nr. 132 din 17 iunie 2016 [citat 07.12.2021] Disponibil: https://www.legis.md/cautare/getResults?doc_ id=128394&lang=ro. A se vedea art. 15 alin. (1) din Legea privind declararea averii și a inte 7. reselor personale nr. 133 din 17 iunie 2016 [citat 07.12.2021] Disponibil: https://www.legis.md/cautare/getResults?doc_id=128397&lang=ro. A se vedea art. 2 alin. (3) lit. b) din Codul administrativ al Republicii Mol 8. dova, nr. 116 din 19 iulie 2018 [citat 07.12.2021] Disponibil: https://www. legis.md/cautare/getResults?doc_id=129135&lang=ro. ”Cu privire la actul de constatare al Autorității Naționale de Integritate în 9. privința doamnei judecător Domnica Manole” [citat 10.01.2022] Disponi bil: https://www.constcourt.md/libview.php?l=ro&idc=7&id=2351&t=/ Media/Noutati/Cu-privire-la-actul-de-constatare-al-Autoritatii-Nationa le-de-Integritate-in-privinta-doamnei-judecator-Domnica-Manole/. .Legea nr. 133/2016 privind declararea averii și a intereselor personale [citat 10. 07.12.2021] Disponibil: https://www.legis.md/cautare/getResults?doc_ id=128397&lang=ro. .Legea nr. 133/2016 privind declararea averii și a intereselor personale [citat 10. 07.12.2021] Disponibil: https://www.legis.md/cautare/getResults?doc_ id=128397&lang=ro. 11. Nota aut.: Similar tuturor autorităților cu funcții de control în stat, noi avem deja precedente de „stat în stat„ cum ar fi în cazul sistemului judecă toresc și al procuraturii. 11. Nota aut.: Similar tuturor autorităților cu funcții de control în stat, noi avem deja precedente de „stat în stat„ cum ar fi în cazul sistemului judecă toresc și al procuraturii. 11. Nota aut.: Similar tuturor autorităților cu funcții de control în stat, noi avem deja precedente de „stat în stat„ cum ar fi în cazul sistemului judecă toresc și al procuraturii. Nota aut.: 12. Referințe bibliografice și resurse Web: Legislația în domeniul integrității nu conține termene destul de concrete și restrânse pentru activitățile de control, lăsând în principal la latitudinea funcționarilor ANI. De exemplu Codul administrativ al RM, nr. 116/2018 (art. 60), prevede un termen general de finalizare a oricărei pro ceduri administrative de 30 de zile, iar legislația în domeniul integrității lărgește procedura respectivă până la ani de zile. A se vedea art. 27-30 din Codul administrativ al Republicii Moldova, nr. 13. 116 din 19 iuie 2018 [citat 10.01.2022] Disponibil: https://www.legis.md/ cautare/getResults?doc_id=129135&lang=ro. A se vedea Capitolul V 14. 1 din Titlul I Cartea a doua din Codul contravențional al Republicii Moldova, nr. 218 din 24 octombrie 2008 [citat 07.12.2021] Dispo nibil: https://www.legis.md/cautare/getResults?doc_id=130231&lang=ro și Capitolul X din Partea generală a Codului penal al Republicii Moldova, nr. 985-XV din 18 aprilie 2002 [citat 07.12.2021] Disponibil: https://www. legis.md/cautare/getResults?doc_id=129474&lang=ro. „EUR-Lex”. Access to European Union law: summaries of EU legislation/ 15. glossary of summaries. [citat 10.01.2022] Disponibil: https://eur-lex.eu ropa.eu/summary/glossary/subsidiarity.html. 384 16. Nota aut.:Prevederea respectivă încalcă procedura intrării în vigoare a ac telor administrative în vigoare care poate fi nu mai devreme de momentul aducerii la cunoștința persoanelor vizate în ele, dar nici cum data emite rii. Cu atât mai mult că conform art. 139 alin.(1) din Codul administra tiv al Republicii Moldova, nr. 116 din 19 iuie 2018, ,,un act administrativ individual devine valabil pentru persoana căreia îi este destinat sau care este afectată de el în momentul în care i se comunică acesteia, cu excepția cazului când actul administrativ însuși stabilește o dată ulterioară pentru obligativitatea sa”. Respectiv termenul de 30 de zile de contestare a actului ANI începe efectiv din data comunicării subiectului declarării, deoarece până la aceea dată actul administrativ individual respectiv nu ar fi valabil sau mai suplu nu ar exista. A se vedea art. 36 din Legea cu privire la Autoritatea Națională de Integri 17. tate, nr. 132 din 17 iunie 2016 [citat 07.12.2021] Disponibil: https://www. legis.md/cautare/getResults?doc_id=128394&lang=ro. A se vedea art. 36 din Legea cu privire la Autoritatea Națională de Integri 17. tate, nr. 132 din 17 iunie 2016 [citat 07.12.2021] Disponibil: https://www. legis.md/cautare/getResults?doc_id=128394&lang=ro. 385 385
https://openalex.org/W4360987789	https://lirias.kuleuven.be/bitstream/20.500.12942/727092/2/fbioe-11-1150170.pdf	English	null	Muscle preflex response to perturbations in locomotion: In vitro experiments and simulations with realistic boundary conditions	Frontiers in bioengineering and biotechnology	2,023	cc-by	13,067	OPEN ACCESS OPEN ACCESS EDITED BY Kamran Iqbal, University of Arkansas at Little Rock, United States REVIEWED BY Reinhard Blickhan, Friedrich Schiller University Jena, Germany Huub Maas, VU Amsterdam, Netherlands CORRESPONDENCE Matthew Araz, matthew.araz@uni-tuebingen.de †Present address: Daniel F. B. Haeuﬂe, Institute of Computer Engineering (ZITI), Heidelberg University, Heidelberg, Germany SPECIALTY SECTION This article was submitted to Biomechanics, a section of the journal Frontiers in Bioengineering and Biotechnology RECEIVED 23 January 2023 ACCEPTED 21 March 2023 PUBLISHED 27 April 2023 CITATION Araz M, Weidner S, Izzi F, Badri-Spröwitz A, Siebert T and Haeuﬂe DFB (2023), Muscle preﬂex response to perturbations in locomotion: In vitro experiments and simulations with realistic boundary conditions. Front. Bioeng. Biotechnol. 11:1150170. doi: 10.3389/fbioe.2023.1150170 OPEN ACCESS EDITED BY Kamran Iqbal, University of Arkansas at Little Rock, United States REVIEWED BY Reinhard Blickhan, Friedrich Schiller University Jena, Germany Huub Maas, VU Amsterdam, Netherlands CORRESPONDENCE Matthew Araz, matthew.araz@uni-tuebingen.de †Present address: Daniel F. B. Haeuﬂe, Institute of Computer Engineering (ZITI), Heidelberg University, Heidelberg, Germany SPECIALTY SECTION This article was submitted to Biomechanics, a section of the journal Frontiers in Bioengineering and Biotechnology RECEIVED 23 January 2023 ACCEPTED 21 March 2023 PUBLISHED 27 April 2023 CITATION Araz M, Weidner S, Izzi F, Badri-Spröwitz A, Siebert T and Haeuﬂe DFB (2023), Muscle preﬂex response to perturbations in locomotion: In vitro experiments and simulations with realistic boundary conditions. Front. Bioeng. Biotechnol. 11:1150170. doi: 10.3389/fbioe.2023.1150170 EDITED BY Kamran Iqbal, University of Arkansas at Little Rock, United States REVIEWED BY Reinhard Blickhan, Friedrich Schiller University Jena, Germany Huub Maas, VU Amsterdam, Netherlands Matthew Araz1,2*, Sven Weidner3, Fabio Izzi1,4, Alexander Badri-Spröwitz4,5, Tobias Siebert3 and Daniel F. B. Haeuﬂe1,2,6† 1Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany, 2Institute for Modelling and Simulation of Biomechanical Systems, University of Stuttgart, Stuttgart, Germany, 3Department of Motion and Exercise Science, Institute of Sport and Movement Science, University of Stuttgart, Stuttgart, Germany, 4Dynamic Locomotion Group, Max Plank Institute for Intelligent Systems, Stuttgart, Germany, 5Department of Mechanical Engineering, KU Leuven, Leuven, Belgium, 6Center for Integrative Neuroscience, University of Tübingen, Tübingen, Germany Neuromuscular control loops feature substantial communication delays, but mammals run robustly even in the most adverse conditions. In vivo experiments and computer simulation results suggest that muscles’ preﬂex—an immediate mechanical response to a perturbation—could be the critical contributor. Muscle preﬂexes act within a few milliseconds, an order of magnitude faster than neural reﬂexes. TYPE Original Research PUBLISHED 27 April 2023 DOI 10.3389/fbioe.2023.1150170 TYPE Original Research PUBLISHED 27 April 2023 DOI 10.3389/fbioe.2023.1150170 TYPE Original Research PUBLISHED 27 April 2023 DOI 10.3389/fbioe.2023.1150170 muscle dynamics, mechanical work, intrinsic properties, stiffness, damping Frontiers in Bioengineering and Biotechnology 1 Introduction We obtained the kinematic trajectories by simulating vertical human hopping driven by a Hill-type muscle model under three levels of perturbations: step-up, no step and step-down. Further, we derived a quasistatic-scenario for muscle ﬁber experiments with the same lengthening patterns over a much larger time to eliminate ﬁber’s velocity effect on the muscle force production. These quasistatic-scenario experiments permit separating the elastic response from the viscous response of muscle ﬁbers. Finally, we compared simulations with muscle ﬁber experiments to test the accuracy of Hill-type models in explaining ﬁber response. Our results show that during the preﬂex phase, intrinsic muscle characteristics adjust the muscle force in response to the perturbation level. Our ﬁndings corroborate that muscle activity can tune mechanical muscle properties in advance. On the other hand, we conﬁrm previous ﬁndings that classical Hill- type muscle models—as the one used in our study—cannot accurately predict the force response in the preﬂex phase. This not only the case within the SRS phase (Hu et al., 2011; De Groote et al., 2017), but also after the SRS, where the change in force with stretch velocity (Weidner et al., 2022) is not predicted by the model. In vivo walking experiments are essential for understanding robust locomotion. However, the functional mechanical and control coupling of muscle groups during whole-body movement complicates unveiling the regulatory principles behind preﬂexes, reﬂexes, and voluntary neuromuscular control. By artiﬁcially contracting individual muscle ﬁbers, in vitro research allows for precise isolation and investigation of muscles’ mechanical properties (Weidner et al., 2022). So far, a wide range of contraction settings have been explored, such as isometric, isotonic, and isovelocity (Brown et al., 2003; Gilliver et al., 2011; Tomalka et al., 2020). Yet, the exact boundary conditions of physiological muscle contraction are hard to replicate in in vitro experiments. Cyclic ﬁber contractions during in vitro experiments and follow-up work loop analyses are relatively realistic (Josephson, 1985). Even though it is possible to extract physiological kinematic trajectories of muscle contraction with the sonomicrometry method during in vivo experiments (Daley et al., 2009; Gordon et al., 2020), extracting the preﬂex phase is still challenging due to the relative oscillations of soft tissue at impact (Christensen et al., 2017). Therefore, stretch- shortening cycle investigations that are limited to sinusoidal length trajectories (Darby et al., 2013) only roughly present locomotion conditions. 1 Introduction ﬁber experiments, mostly at maximum activity, which are controlled experimental conditions differing greatly from in vivo muscle loading. Compared to data from gait recordings, Hill-type muscle models were inaccurate in predicting muscle force during high-speed locomotion (Lee et al., 2013; Dick et al., 2017). Furthermore, several studies showed that Hill-type muscle models can only predict accurate joint kinetics and kinematics for perturbed quiet stance (Hu et al., 2011; De Groote et al., 2017) if they consider a model extension accounting for short-range stiffness (Cui et al., 2008). Therefore, it still needs to be discovered to what extent simulation studies with Hill-type models can validate experimental research on muscle preﬂex. On the other hand, in vitro experiments are required to test individual muscle ﬁbers’ response to unexpected perturbation. Legged locomotion on uneven terrain is a complex motor control task performed seemingly effortless by humans and other animals (Blickhan et al., 2007). Animals reject unexpected ground perturbations (Daley and Biewener, 2006; Müller et al., 2014), despite considerable sensorimotor transmission delays affecting the feedback control (More et al., 2010; More and Donelan, 2018). This ability has long puzzled researchers in biomechanics and motorcontrol science. In vivo research on perturbed legged locomotion suggests that the intrinsic mechanical properties of muscles are essential for dynamic stability during the ﬁrst 30 ms–50 ms after touchdown (Nishikawa et al., 2007; Daley et al., 2009; Gordon et al., 2020). During this brief interval, muscles and tendons react instantly through elastic and viscous- like properties. Brown and Loeb (2000, p. 161) labeled it preﬂex; the “. . .zero-delay, intrinsic response of a neuromusculoskeletal system to a perturbation”. This study aims at understanding how individual muscle ﬁbers exploit their intrinsic mechanical properties to respond to perturbations in realistic settings (in terms of physiological boundary conditions). We focus on how muscles’ elastic and viscous properties regulate energy absorption during the preﬂex phase to reject perturbations during locomotion impacts. We hypothesize that (1) the muscle’s force and mechanical work during and after the short-range stiffness (SRS) period, but within the 30 ms preﬂex period, changes in reaction to differences in stretch velocities induced by step-up and step- down ground level perturbations, and (2) mechanical muscle properties can be tuned by changing activity level in advance to touchdown. We conducted muscle ﬁber experiments with realistic kinematic trajectories at three different activity levels to prove our hypotheses. OPEN ACCESS Their short-lasting action makes mechanical preﬂexes hard to quantify in vivo. Muscle models, on the other hand, require further improvement of their prediction accuracy during the non-standard conditions of perturbed locomotion. Our study aims to quantify the mechanical work done by muscles during the preﬂex phase (preﬂex work) and test their mechanical force modulation. We performed in vitro experiments with biological muscle ﬁbers under physiological boundary conditions, which we determined in computer simulations of perturbed hopping. Our ﬁndings show that muscles initially resist impacts with a stereotypical stiffness response—identiﬁed as short-range stiffness—regardless of the exact perturbation condition. We then observe a velocity adaptation to the force related to the amount of perturbation similar to a damping response. The main contributor to the preﬂex work modulation is not the change in force due to a change in ﬁber stretch velocity (ﬁber damping characteristics) but the change in magnitude of the stretch due to the leg dynamics in the perturbed conditions. Our results conﬁrm previous ﬁndings that muscle stiffness is activity-dependent and show that also damping characteristics are activity-dependent. These results indicate that neural control could tune the preﬂex properties of muscles in expectation of ground conditions leading to previously inexplicable neuromuscular adaptation speeds. CITATION Araz M, Weidner S, Izzi F, Badri-Spröwitz A, Siebert T and Haeuﬂe DFB (2023), Muscle preﬂex response to perturbations in locomotion: In vitro experiments and simulations with realistic boundary conditions. Front. Bioeng. Biotechnol. 11:1150170. doi: 10.3389/fbioe.2023.1150170 COPYRIGHT © 2023 Araz, Weidner, Izzi, Badri- Spröwitz, Siebert and Haeuﬂe. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 01 Frontiers in Bioengineering and Biotechnology frontiersin.org Frontiers in Bioengineering and Biotechnology Araz et al. 10.3389/fbioe.2023.1150170 10.3389/fbioe.2023.1150170 1 Introduction Previous simulation studies support the hypothesis that intrinsic muscle properties play a crucial role in stabilizing locomotion against disturbances (Gerritsen et al., 1998; Van Der Krogt et al., 2009; John et al., 2013). Simulation studies revealed that feedforward adjustment of muscle stimulation, as observed during human locomotion (Müller et al., 2015), may allow to adjust muscle mechanics according to perturbed impact conditions (Haeuﬂe et al., 2010; 2018). As a means of investigation, computer simulations combine the advantages of in vivo and in vitro investigations. They enable the analysis of complex whole-body movements similar to in vivo research while providing access to difﬁcult-to-measure variables, similar to in vitro experiments. Nevertheless, computer models depend on simpliﬁed assumptions. Most investigations of muscle preﬂex use classic Hill-type muscle models, which are restricted in their ability to describe muscle contraction outside of speciﬁc conditions (Siebert et al., 2021). Hill-type muscle models are parameterized with empirical data from isometric, isotonic, and isovelocity muscle Frontiers in Bioengineering and Biotechnology frontiersin.org 2 Materials and methods However, the data after the preﬂex is measured 2 Materials and methods The goal of this study was to test the force response of muscle ﬁbers in realistic perturbation scenarios. Boundary conditions for in vitro muscle ﬁber experiments were derived from a human hopping simulation (Figure 1A). It is a simpliﬁed model of human hopping which consist of a point mass, a single leg with 02 frontiersin.org 10.3389/fbioe.2023.1150170 Araz et al. FIGURE 1 Simulations and in vitro experiments applied in this study. A Hill-type model muscle-tendon unit drives a knee extensor unit of the hopper (Geyer et al., 2003; Haeuﬂe et al., 2014; Izzi et al., 2023). Single-leg hopping is computer-simulated (A) with three perturbation scenarios: 5 cm step-up (P↑), no perturbation (P0), and 5 cm step-down (P↓). The model outputs are length changes of the contractile element (lCE, (B)), and contraction velocities (_lCE, (B)). One hopping cycle from touchdown (TD) to toe-off (TO) is extracted for the analysis. Since the focus of this study is the muscle behavior during the preﬂex phase of a hopping cycle (the ﬁrst 30 ms of the stance phase), we focused on time zone shown with the blue area (B). In this time zone there is no inﬂuence of reﬂexes on the muscle activation and we assume no activity rise due to feed-forward muscle activation. Thus, constant stimulation proﬁle (uCE-orange line is shown only for 15% stimulation level, (B)) is then applied to in vitro muscle ﬁber experiments (C) and isolated contractile element (CE) simulations (D). Constant stimulation in experiments and isolated contractile element simulations were used since the in vitro setup does not allow changing stimulation levels within a stretch-shortening cycle. Since the muscle ﬁbres used in in vitro experiments (C) are isolated from the tendon, the contractile element (CE) and parallel elastic element (PEE) of the Hill-type muscle model are also isolated from the tendon unit (D). FIGURE 1 Simulations and in vitro experiments applied in this study. A Hill-type model muscle-tendon unit drives a knee extensor unit of the hopper (Geyer et al., 2003; Haeuﬂe et al., 2014; Izzi et al., 2023). Single-leg hopping is computer-simulated (A) with three perturbation scenarios: 5 cm step-up (P↑), no perturbation (P0), and 5 cm step-down (P↓). The model outputs are length changes of the contractile element (lCE, (B)), and contraction velocities (_lCE, (B)). One hopping cycle from touchdown (TD) to toe-off (TO) is extracted for the analysis. 2 Materials and methods Since the focus of this study is the muscle behavior during the preﬂex phase of a hopping cycle (the ﬁrst 30 ms of the stance phase), we focused on time zone shown with the blue area (B). In this time zone there is no inﬂuence of reﬂexes on the muscle activation and we assume no activity rise due to feed-forward muscle activation. Thus, constant stimulation proﬁle (uCE-orange line is shown only for 15% stimulation level, (B)) is then applied to in vitro muscle ﬁber experiments (C) and isolated contractile element (CE) simulations (D). Constant stimulation in experiments and isolated contractile element simulations were used since the in vitro setup does not allow changing stimulation levels within a stretch-shortening cycle. Since the muscle ﬁbres used in in vitro experiments (C) are isolated from the tendon, the contractile element (CE) and parallel elastic element (PEE) of the Hill-type muscle model are also isolated from the tendon unit (D). two segments, and a classical Hill-type muscle model connected as a knee extensor (Geyer et al., 2003; Haeuﬂe et al., 2014; Izzi et al., 2023) (For details of the model see Supplementary Section S1). We performed three simulations with the hopping model: a no- perturbation reference hopping (P0), a step-up perturbation (P↑), and a step-down perturbation (P↓). Figure 1B shows the stimulation proﬁle applied during the hopping simulation. During the ﬁrst 30 ms (preﬂex), the stimulation is kept constant due to the delay of neural transmission, and response against the perturbation is dependent only on elastic and viscous intrinsic properties. Then, stimulation rises linearly with time (approximated from experimental ﬁndings for hopping (Moritz and Farley, 2004) and walking (Müller et al., 2015; 2020)). We extracted kinematic trajectories and the stimulation state during the preﬂex phase (Figure 1B) of the contractile element. These data were used as boundary conditions for muscle ﬁber experiments (conducted with the ﬁbers taken from rat muscles) and their corresponding simulations of the isolated contractile element (Figures 1C, D). Notice that in the vertical hopping model (Figure 1A) the neural stimulation (u) is ﬁrst ﬁltered by activation dynamics (Rockenfeller et al., 2015; Hatze, 1977). However, in the simulation of isolated contractile element there is no activation dynamics included, the neural stimulation sent to the muscle and the muscle activation level is equal (u = a). Frontiers in Bioengineering and Biotechnology 2 Materials and methods This was necessary since the simulation of the isolated contractile element mimics the in vitro muscle ﬁber experiments for which no activation dynamics take place. The highlighted preﬂex phase (Figure 1B, shaded area)—is the focus of our study. Figure 1B shows also the behavior shortly after the preﬂex phase. However, the data after the preﬂex is measured with constant activity levels, as the in vitro setup does not allow for a time-controlled activity change. In the hopping simulation, the muscle activity rises after the preﬂex phase. We recorded force-length traces during these experiments and matching simulations and analyzed the preﬂex phases of work- loops from the mechanical work of the muscle ﬁber. The following sections provide details of the experiments and simulations conducted. two segments, and a classical Hill-type muscle model connected as a knee extensor (Geyer et al., 2003; Haeuﬂe et al., 2014; Izzi et al., 2023) (For details of the model see Supplementary Section S1). We performed three simulations with the hopping model: a no- perturbation reference hopping (P0), a step-up perturbation (P↑), and a step-down perturbation (P↓). Figure 1B shows the stimulation proﬁle applied during the hopping simulation. During the ﬁrst 30 ms (preﬂex), the stimulation is kept constant due to the delay of neural transmission, and response against the perturbation is dependent only on elastic and viscous intrinsic properties. Then, stimulation rises linearly with time (approximated from experimental ﬁndings for hopping (Moritz and Farley, 2004) and walking (Müller et al., 2015; 2020)). We extracted kinematic trajectories and the stimulation state during the preﬂex phase (Figure 1B) of the contractile element. These data were used as boundary conditions for muscle ﬁber experiments (conducted with the ﬁbers taken from rat muscles) and their corresponding simulations of the isolated contractile element (Figures 1C, D). Notice that in the vertical hopping model (Figure 1A) the neural stimulation (u) is ﬁrst ﬁltered by activation dynamics (Rockenfeller et al., 2015; Hatze, 1977). However, in the simulation of isolated contractile element there is no activation dynamics included, the neural stimulation sent to the muscle and the muscle activation level is equal (u = a). This was necessary since the simulation of the isolated contractile element mimics the in vitro muscle ﬁber experiments for which no activation dynamics take place. The highlighted preﬂex phase (Figure 1B, shaded area)—is the focus of our study. Figure 1B shows also the behavior shortly after the preﬂex phase. frontiersin.org 2.1.2 Experimental setup The skinned muscle ﬁber was transferred from the skinning solution to the experimental chamber of the ﬁber test apparatus (600A, Aurora Scientiﬁc, ON, Canada). One clipped end was attached to a length controller (model 308B, Aurora Scientiﬁc, ON, Canada) and the other end to a force transducer (model 403A, Aurora Scientiﬁc, ON, Canada). Both attached ends were ﬁxed with ﬁngernail polish diluted with acetone (Getz et al., 1998). Transitions from the ﬁber end to the clip were treated with glutaraldehyde in rigor solution to improve mechanical performance and stability during the experiment (Hilber and Galler, 1998). The central ﬁber segment was focused in the microscope and used to optically measure the sarcomere length (Weidner et al., 2022), which was set to 2.5 µm (means ± standard deviation) in the beginning. At this optimal sarcomere length lopt the ﬁber produces its maximum force Fmax (Stephenson and Williams, 1982). The corresponding muscle ﬁber length is deﬁned as lopt. The height (h) and width (w) of the ﬁber was measured in 0.1 mm increments over the entire length of the ﬁber using a 10 x extra long working distance dry lens (NA 0.60, Nikon, Japan) and a 10 x eyepiece. The cross- sectional area of all tested muscle ﬁbers was determined 5.25 × 10−9 m2 (±1.5 × 10−9) assuming an elliptical cross-section (π × h × w/4). The in vitro experiment included isometric contractions at optimal ﬁber length and three hopping stretch-shortening cycles based on the simulation data of the hopping model (Section 2.2). First, the activity level of the ﬁber in three sub-maximal conditions was checked using isometric contractions in 5%, 15%, 25%, and supra maximal activation solution at optimal ﬁber length. This step ensured matching boundary conditions with the simulation data. A ﬂow chart of an experimental day for a single ﬁber is shown in Supplementary Figure S3. Hopping stretch-shortening cycles were applied to the ﬁber according to length and velocity data extracted from the simulations in the simulated dynamic-scenarios, and modiﬁed quasistatic-scenarios (see Section 2.2 for more details). A high-speed video system (Aurora Scientiﬁc, 901B, Canada) in combination with a 10 x extra long working distance dry objective (NA 0.40, Nikon, Japan) and an accessory lens (2.5 x, Nikon, Japan) visualized and tracked dynamic changes in the sarcomere length. Videos were recorded at 300 Hz recording frequency. 2.1.2 Experimental setup Both the order of stretch-shortening proﬁles and the order of activity levels within a perturbation, called a “block”, were randomized. Each block was surrounded by isometric reference contractions at optimal ﬁber length and full activity to take into account ﬁber degradation during force data normalization (Supplementary Figure S3). 2.1.1 Fiber preparation One M. extensor digitorum longus (EDL) was extracted from a single female Wistar rat, which was sacriﬁced with an overdose of CO2 shortly before. We used n = 9 ﬁbers from the extracted EDL muscle for our experiment. The specimen’s age was 8–10 months, at a body weight of 300 g–350 g. The specimen was kept at a 12 h light and 12 h dark cycle at a housing temperature of 22°C. The EDL muscle was obtained from the left hind limb. The experiment was conducted according to the guidelines of ARRIVE and approved according to the German animal protection law (Tierschutzgesetz §4(3), permit no. T170/18ST). The techniques used for muscle preparation, storage, and activation of skinned single muscle ﬁbers were carried out as described in detail in Tomalka et al. (2017). In summary, the EDL was prepared in 6–8 small ﬁber bundles, which were permeabilized in a skinning solution (see “Solutions”; Section 2.1.3) at 4°C temperature directly after preparation. Fiber bundles were transferred to a storage solution made of 50% glycerol and 50% 03 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 GLH (pCa 9.0). The preactivating solution contained 0.1 mol TES, 6.93 mmol MgCl2, 5.45 mmol Na2ATP, 0.1 mmol EGTA, 19.49 mmol Na2CP, 10 mmol GLH, and 24.9 mmol HDTA. The skinning solution contained 0.17 mol potassium propionate, 2.5 mmol MgCl2, 2.5 mmol Na2ATP, 5 mmol EGTA, 10 mmol IMID, and 0.2 mmol PMSF. Recipes for activation solutions (‘ACT’) are shown in Table 1. The storage solution is the same as the skinning solution, except for the presence of 10 mmol GLH and 50% glycerol vol/vol. Cysteine and cysteine/serine protease inhibitors [trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, E−64, 10 mM; leupeptin, 20 mg/mL] were added to all solutions to preserve lattice proteins and thus sarcomere homogeneity (Linari et al., 2007; Tomalka et al., 2017). KOH was applied to adjust to a pH 7.1 at 12 °C. Then, 450 U/mL of creatine kinase were added to all except skinning and storage solutions. Creatine kinase was obtained from Roche, Mannheim, Germany, and the remaining chemicals were obtained from Sigma, St Louis, MO. According to our calibration curve (Supplementary Figure S2) we chose concentrations of 6.73 pCa, 6.34 pCa and 6.3 pCa to best match the simulations boundary conditions. GLH (pCa 9.0). The preactivating solution contained 0.1 mol TES, 6.93 mmol MgCl2, 5.45 mmol Na2ATP, 0.1 mmol EGTA, 19.49 mmol Na2CP, 10 mmol GLH, and 24.9 mmol HDTA. 2.1.4 Experimental protocol All experimental trials were conducted at a solution temperature of 12°C (±0.1). At this temperature, the skinned muscle ﬁbers proved stable during work-loop experiments (Tomalka et al., 2021; Tomalka et al., 2020). Fibers can tolerate activation and active stretch protocols over a long period (Ranatunga, 1982; Ranatunga, 1984). A three-step approach was used to activate the ﬁbers by calcium diffusion. First, muscle ﬁbers were immersed for 60 s in a preactivation solution for equilibration. The ﬁber was then transferred to the activation solution. This led to a rapid increase in force until a plateau was reached. We deﬁned the plateau as an isometric force increase of less than 1% rise of force within 1.5 s. After reaching the plateau, the perturbation was carried out. In the last step, the ﬁber was transferred to the relaxing solution, in which it was prepared for the subsequent activation for 400 s using cycling protocols (Tomalka et al., 2017). 2.1.1 Fiber preparation The skinning solution contained 0.17 mol potassium propionate, 2.5 mmol MgCl2, 2.5 mmol Na2ATP, 5 mmol EGTA, 10 mmol IMID, and 0.2 mmol PMSF. Recipes for activation solutions (‘ACT’) are shown in Table 1. The storage solution is the same as the skinning solution, except for the presence of 10 mmol GLH and 50% glycerol vol/vol. Cysteine and cysteine/serine protease inhibitors [trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane, E−64, 10 mM; leupeptin, 20 mg/mL] were added to all solutions to preserve lattice proteins and thus sarcomere homogeneity (Linari et al., 2007; Tomalka et al., 2017). KOH was applied to adjust to a pH 7.1 at 12 °C. Then, 450 U/mL of creatine kinase were added to all except skinning and storage solutions. Creatine kinase was obtained from Roche, Mannheim, Germany, and the remaining chemicals were obtained from Sigma, St Louis, MO. According to our calibration curve (Supplementary Figure S2) we chose concentrations of 6.73 pCa, 6.34 pCa and 6.3 pCa to best match the simulations boundary conditions. TABLE 1 Recipe of activation solutions used, values are in [mmol]. TABLE 1 Recipe of activation solutions used, values are in [mmol]. 5% ACT 15% ACT 25% ACT 100% ACT TES 100.000 100.000 100.000 100.000 MgCl2 7.183 6.995 6.980 6.760 EGTA 11.250 6.250 5.852 0.000 CaEGTA 13.750 18.750 19.147 25.000 Na2ATP 5.451 5.455 5.455 5.460 KPi 0 0 0 0 Na2CP 19.319 19.395 19.401 19.490 GSH 10.000 10.000 10.000 10.000 skinning solution, and kept at −22°C for 6–8 weeks. Prior to conducting experiments, ﬁbers were removed from the bundle using a dissecting microscope and ﬁne forceps. Single ﬁbers were cut into smaller muscle ﬁber segments with a length of 1 mm. Aluminum T-shaped clips were folded around both ends of the ﬁber. The ﬁber was then treated with a skinning solution consisting of a relaxing solution with 1% vol/vol Triton-X 100 for 3 min at 4°C until the complete removal of internal ﬁber membranes (Linari et al., 2007). Frontiers in Bioengineering and Biotechnology frontiersin.org 2.2.2 Extracting boundary conditions We simulated hopping for no-perturbation locomotion on ground level (P0), 5 cm step-up perturbation (P↑), and 5 cm step-down perturbation (P↓). Beyond step-up perturbations of 5 cm, the single- leg hopper generates unstable hopping patterns. Thus we decided to use a maximum perturbation height of 5 cm. The contractile element length and velocity proﬁles were extracted for each perturbation level. These kinematic data were used in muscle ﬁber experiments and isolated contractile element simulations to compare their reactions to the perturbations during the preﬂex phase. We further derived quasistatic-scenario boundary conditions to differentiate between the muscle ﬁbers’ velocity-dependent and length- dependent force modulation. To create a length-dependent force modulation, we generated quasistatic-scenario boundary conditions for each perturbation level. In these conditions, the time duration of the contractile element lengthening proﬁles obtained from each perturbation level was expanded by 80 times compared to the original duration. Thereby, the contraction velocity was decreased to negligible levels without exceeding the minimum speed limits of the experimental setup. Hence, the viscous contribution was minimized from the muscle ﬁber force response, and the muscles only reacted with their elastic properties to the perturbations. FCE,e _lCE > 0 Fmax aFisom + Arel,e 1 − _lCE Brel,elopt −Arel,e ⎛ ⎜ ⎜ ⎝ ⎞⎟⎟⎠ (1) FCE,e _lCE > 0 Fmax aFisom + Arel,e 1 − _lCE Brel,elopt −Arel,e ⎛ ⎜ ⎜ ⎝ ⎞⎟⎟⎠ (1) (1) Here _lCE is the ﬁber contraction velocity, Fmax is the maximum isometric force that the contractile element can generate, Fisom is the isometric force that the contractile element generates according to the current muscle length, Arel,e and Brel,e are the normalized Hill parameters for the eccentric phase, and a is the activity level. Figure 2 shows force-velocity traces predicted by the Hill-type muscle model for sub-maximal (5%, 15% and 25%), and full activity (100%). 2.1.3 Solutions The relaxing solution contained 0.1 mol TES, 7.7 mmol MgCl2, 5.44 mmol Na2ATP, 25 mmol EGTA, 19.11 mmol Na2CP, 10 mmol Velocity, force, and length data were recorded at 1 kHz for isometric and quasistatic-scenario trials and 10 kHz for high-speed 04 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 trials with an A/D interface (604A, Aurora Scientiﬁc, ON, Canada). The data acquisition was carried out with real-time software (600A, Aurora Scientiﬁc, ON, Canada). Data were loaded into MATLAB (MathWorks, MA, United States) and analyzed with a custom- written script. Forces during perturbation trials for every single ﬁber were divided by individual Fmax, and likewise, ﬁber length l by individual lopt, to normalize them. FIGURE 2 Force-velocity relation for the contractile element for activity levels of 5%, 15%, 25% and 100%. Shown intervals for each perturbation represent the stretch velocity ranges from touchdown to the end of preﬂex phase. Frontiers in Bioengineering and Biotechnology 2.2.1 Generating hopping trajectories in simulation 2.2.1 Generating hopping trajectories in simulation To identify realistic boundary conditions for in vitro muscle ﬁber experiments, we extracted contractile element kinematics from a single-leg hopper simulation [(Izzi et al., 2023) based on the model by Geyer et al. (2003)]. The single-leg hopper is driven by a Hill-type muscle-tendon unit (MTU) model. The MTU model considers four elements: a contractile element representing the muscle ﬁbers, a parallel elastic (PEE), a serial elastic (SEE), and a series damping (SDE) element (Haeuﬂe et al., 2014). The modeled muscle-tendon unit extends the knee joint (Figure 1A). The leg features two massless segments connected by the knee hinge joint. The body mass is represented as a point mass located at the hip joint. For further details on the model, see Supplementary Material Section S1. FIGURE 2 Force-velocity relation for the contractile element for activity levels of 5%, 15%, 25% and 100%. Shown intervals for each perturbation represent the stretch velocity ranges from touchdown to the end of preﬂex phase. (MATLAB 2021b). All other parameters are based on Bayer et al. (2017), Kistemaker et al. (2006) and Mörl et al. (2012) and tabulated in the Supplementary Table S1. We simulated stable periodic hopping with the hopper model and introduced a step-up and a step-down perturbation (Figure 1A). During the ﬂight phase, the muscle was stimulated with 15% constant stimulation, and the knee joint was ﬁxed. After touchdown, the constant stimulation level continued for 30 ms throughout the preﬂex phase and then increased with a ramp input (b = 10 s−1, Figure 1B). Despite the constant stimulation during the preﬂex phase, the contractile element can change its force due to the elastic and viscous intrinsic properties, which are related to force-length and force-velocity, respectively. Since the Hill-type muscle is extending the knee in the hopping simulation, muscle-tendon unit and contractile element are stretched at the initial phases of the stretch-shortening cycle. Thus, the model operates at the eccentric section of the force-velocity relation (Haeuﬂe et al., 2014): 2.3.2 Statistics In the dynamic-scenario analysis, the muscle ﬁbers’ response is a combination of two mechanical features: elasticity and viscosity. To identify their individual contributions, we minimized the parameter responsible for the viscous contribution—the stretch velocity. We performed quasistatic-scenario experiments, where muscles were stretched with the same lengthening proﬁles as in dynamic-scenario experiments, but at super-low velocities. Hence, with this experimental design, we expect to see only the elastic muscle ﬁber response. Still, even at negligible stretch velocities, we observed a similar preﬂex work trend between perturbation levels in quasistatic-scenario experiments and dynamic-scenario experiments (Figures 4A, B). SPSS 27 (IBM Corp., Armonk, NY) was used for the statistical analysis, with a signiﬁcance level of p = 0.05. Initially, we tested for normal data distribution by running a Shapiro-Wilk, which was negative. Hence, we used a Friedman test to elucidate differences between the applied perturbations within one activity level. We executed comparisons pairwise for post hoc experimental data analysis. Results were fed into a Bonferroni correction to take multiple testing into account. We tested for differences between similar activity levels and applied perturbation between dynamic-scenario and quasistatic-scenario conditions with a two-sample paired sign test. Effect sizes for the pairwise comparisons were classiﬁed as small (d < 0.3), medium (0.3 < d < 0.5), and large (d > 0.5) using Cohen’s d (Cohen, 2013). Surprisingly, the Hill-type muscle model predicted the amount of preﬂex work accurately for the dynamic-scenario experiments (Figures 4A, C) and the quasistatic-scenario experiments (Figures 4B, D). Such correct prediction is an expected outcome for quasistatic-scenario experiments but not for dynamic-scenario experiments, as the model’s dynamics differed compared to the muscle ﬁber dynamics (Figures 3A, C). 2.2.3 Simulating isolated contractile element response The response we observed from the skinned ﬁber experiments does not match with the prediction of the isolated contractile element of the Hill-type muscle model (Figures 3A, C). For the Hill-type muscle model, an effect of the perturbation is only observed at touchdown (0.56 ~lCE, Figure 3C). Right after touchdown, the response of the Hill-type muscle model reaches the same force level regardless of the perturbation state and then increases with the same linear trend. Therefore, the model did not predict the modulation in the muscle’s force response due to the perturbation. Contrary to the dynamic-scenario experiments (Figure 3A), force responses of skinned ﬁbers in quasistatic-scenario stretches do not change according to the perturbation level (Figure 3B), during preﬂex. Initial force and the rise in force are similar for all perturbation levels. This result matches the prediction of the isolated contractile element of the Hill-type muscle model (Figure 3D). The response we observed from the skinned ﬁber experiments does not match with the prediction of the isolated contractile element of the Hill-type muscle model (Figures 3A, C). For the Hill-type muscle model, an effect of the perturbation is only observed at touchdown (0.56 ~lCE, Figure 3C). Right after touchdown, the response of the Hill-type muscle model reaches the same force level regardless of the perturbation state and then increases with the same linear trend. Therefore, the model did not predict the modulation in the muscle’s force response due to the perturbation. Contrary to the dynamic-scenario experiments (Figure 3A), force responses of skinned ﬁbers in quasistatic-scenario stretches do not change according to the perturbation level (Figure 3B), during preﬂex. Initial force and the rise in force are similar for all perturbation levels. This result matches the prediction of the isolated contractile element of the Hill-type muscle model (Figure 3D). 2.3.1 Data analysis We found that intrinsic muscle properties adjust the mechanical work during the preﬂex phase (preﬂex work) according to the perturbation condition. This is true in dynamic-scenario and quasistatic-scenario tests, both during experiments and simulation, and for all activity levels (Figure 4). The analysis of stretch-shortening cycles of both experimental and simulated ﬁber contractions focused on the preﬂex phase, which is the ﬁrst 30 ms of dynamic-scenarios. We analyzed data in quasistatic- scenarios condition until the ﬁber lengthening reached the same level as at the end of the preﬂex phase in the dynamic-scenario conditions. We calculated the areas under force-length curves with the trapz function (MATLAB 2021b) to measure the work done by the muscle ﬁbers and the isolated contractile element model. In addition, we estimated the muscle ﬁber’s short-range stiffness from the slope of ﬁtted force-length curves during the initial phase of preﬂex [from 0.57–0.59~lCE (lCE/lopt)]. This initial phase begins where muscles start to generate force (≈0.57~lCE) and ends where the force responses start to deviate due to the stretch velocities (≈0.59~lCE). Then, to observe the effect of velocity on stiffness, we calculated the stiffness during the quasistatic-scenario stretch for the same boundary conditions. The preﬂex work increases when comparing the step-up to the step-down perturbation, according to the dynamic-scenario analysis of skinned ﬁbers (Figure 4A). Albeit no signiﬁcant difference among perturbation states at 5% activity level (p = 0.169), a perturbation inﬂuence on preﬂex work is observable for 15% (χ2 = 12.61; p = 0.002) and 25% (χ2 = 14; p = 0.001) activity levels (Supplementary Table S2). Preﬂex work changes signiﬁcantly between activity levels. For the same kinematic proﬁles, the work done by skinned ﬁbers increases if they are activated more (5%–25% activity level; p = 0.001). The work differences between the perturbation conditions increase with an increase in activity level. See the supplementary materials (Supplementary Table S3) for further details. Frontiers in Bioengineering and Biotechnology 2.2.3 Simulating isolated contractile element response In the hopping simulation, the Hill-type muscle model calculates contractile element kinematics according to the dynamic balance of the serial (SEE and SDE) and contractile (CE and PEE) side of the model. However, in vitro experiments are conducted only with isolated muscle fascicles. To match in vitro conditions, we ran simulations solely with an isolated contractile element. Thereby, For a comparison between the biological muscle ﬁber and the Hill-type muscle model behavior, the parameters of the isometric force-length curve of the model were optimized to ﬁt experimental data (Stephenson, 2003). More precisely, the width of the normalized bell-curve ΔWlimb and its exponent ]CE,limb of the ascending limb were optimized with the lsqcurveﬁt function 05 frontiersin.org 10.3389/fbioe.2023.1150170 10.3389/fbioe.2023.1150170 Araz et al. the isolated responses of the CE—corresponding to the muscle ﬁbers—were calculated according to the provided contractile element length (lCE), contraction velocity (_lCE) and activity (a): within the preﬂex phase (Figure 3A, thick lines). Work loops of dynamic-scenario experiments for skinned ﬁbers show muscle ﬁbers are initially responding with similar force and with a linear and increasing trend between touchdown and 0.59 ~lCE in all perturbations (Figure 3A). After 0.59 ~lCE, the force differs depending on the perturbation level, i.e., force is highest in the step-down perturbation (Figure 3A). within the preﬂex phase (Figure 3A, thick lines). Work loops of dynamic-scenario experiments for skinned ﬁbers show muscle ﬁbers are initially responding with similar force and with a linear and increasing trend between touchdown and 0.59 ~lCE in all perturbations (Figure 3A). After 0.59 ~lCE, the force differs depending on the perturbation level, i.e., force is highest in the step-down perturbation (Figure 3A). FCE f lCE, _lCE, a (2) (2) We obtained the kinematic data of the contractile element from hopping simulations for step-up, no step and step-down perturbations. All perturbation cases were applied as input to the isolated contractile element model with constant activity levels of 5%, 15%, and 25%. Although the activity level increases after the preﬂex phase during hopping simulations, isolated contractile element model simulations must be kept constant to reproduce the conditions of in vitro muscle ﬁber experiments. In the experimental setup, the stimulation level is arranged with chemical baths, as explained in Section 2.1.3. The setup allows only a single stimulation level for each stretch-shortening cycle. Therefore, stimulation levels were kept constant in isolated contractile element simulations to match the experimental in vitro conditions. 3 Results During in vitro experiments, we found that intrinsic muscle properties adjust the force response to the perturbation condition 06 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 FIGURE 3 Shifted work loops for dynamic-scenario and quasistatic-scenario analysis step-up (P↑), no (P0) and step-down (P↓) perturbations for both experiments (A,B) and simulations (C,D) at 15% activity level (work loops for 5% and 25% are shared in Supplementary Figures S4, S5, respectively). The experimental data presented on (A,B) show the mean of all experimental trials. From touchdown to toe-off, all stretch-shortening cycle loops are plotted in the clockwise direction, and the thick and thin sections of the loops represent the preﬂex and remaining part of the stretch-shortening cycle, respectively. The preﬂex stretch gets longer from step-up to step-down perturbation since the muscle stretches faster in the same amount of time. The force-time curves for all experimental conditions with standard deviations can be found in Supplementary Figure S6 and Supplementary Figure S7 for dynamic-scenario and quasistatic-scenario, respectively. FIGURE 3 Shifted work loops for dynamic-scenario and quasistatic-scenario analysis step-up (P↑), no (P0) and step-down (P↓) perturbations for both experiments (A,B) and simulations (C,D) at 15% activity level (work loops for 5% and 25% are shared in Supplementary Figures S4, S5, respectively). The experimental data presented on (A,B) show the mean of all experimental trials. From touchdown to toe-off, all stretch-shortening cycle loops are plotted in the clockwise direction, and the thick and thin sections of the loops represent the preﬂex and remaining part of the stretch-shortening cycle, respectively. The preﬂex stretch gets longer from step-up to step-down perturbation since the muscle stretches faster in the same amount of time. The force-time curves for all experimental conditions with standard deviations can be found in Supplementary Figure S6 and Supplementary Figure S7 for dynamic-scenario and quasistatic-scenario, respectively. Work loops obtained from the dynamic-scenario analysis (Figure 3A; Supplementary Figure S4A; Supplementary Figure S5A) show that the force response of the muscle ﬁbers is almost identical within the short-range stiffness (Rack and Westbury, 1974) regardless of the velocity proﬁle. The force-time curves of all experimental conditions with standard deviations can be found in Supplementary Figure S6; Supplementary Figure S7 for the dynamic- and quasistatic-scenarios, respectively. Only after the short-range stiffness phase, the force and energy are affected by velocity (Figure 5). Frontiers in Bioengineering and Biotechnology 3 Results To understand the inﬂuence of velocity on the preﬂex work, we measure the area after the end of the short-range stiffness phase until the end of preﬂex, for the step-up perturbation condition (Figure 5A, inset: shaded areas). Work done in this phase is slightly higher for the faster stretches at 15% and 25% activity levels. However, the differences between the perturbation levels are not signiﬁcant (15% activity: χ2 = 4.67, p = 0.097; 25% activity: χ2 = 3.56, p = 0.167; See Supplementary Table S2 for more details). dynamic-scenario and quasistatic-scenario experiments are almost identical, and we measured no signiﬁcant effect of the velocity on the preﬂex work (Supplementary Table S4). Both comparisons between perturbations and between dynamic- and quasistatic-scenarios showed that velocity differences had no signiﬁcant effect on the preﬂex work. Analysis of the short-range stiffness shows no difference between perturbations but signiﬁcant differences between activity levels (Figure 6A). In quasistatic-scenario experiments, we found no signiﬁcant differences between perturbation levels. However, short- range stiffness was less in quasistatic-scenario stretches (Figure 6B) than in dynamic-scenario experiments (Figure 6A), and the difference between them increased with higher activities (Supplementary Table S4). Hence, short-range stiffness is increasing from quasistatic-scenario (Figure 6D) to dynamic- scenario velocities (Figure 6C). However, short-range stiffness does not change according to the difference in velocity between the perturbation levels (Figure 6C, different shades of thick blue lines). Besides, the stiffness value can be arranged by changing the activity level. We compared activity levels affecting muscle work to see whether muscles’ viscous properties are tunable. Our results show that the activity level inﬂuences the amount of viscous contribution (Figure 5A) similar to the preﬂex work (Figure 4A). For the same kinematic proﬁles, a rising activity level causes a work increase by viscous characteristics of muscle ﬁbers (15%–25% activity level; p = 0.001). frontiersin.org 4 Discussion To understand the velocity-related adaptation throughout the preﬂex phase, we subtract the work done in the quasistatic-scenario experiments from dynamic-scenario experiments, shown as inset in Figure 5B. Surprisingly, the amount of work done by muscle ﬁbers at In this study, we presented the ﬁrst in vitro experiments conducted under realistic boundary conditions and activity levels of perturbed hopping. Our aim is to understand how intrinsic 07 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 FIGURE 4 The amount of preﬂex work done by skinned ﬁbers and the isolated contractile element of the Hill-type muscle model for all perturbation states and activity levels. In the dynamic-scenario analysis, work done during the preﬂex phase was calculated as the area under the force curve. Shaded areas in the insets (A) and (B) indicate changing perturbation levels. In the quasistatic-scenario analysis, the area till the lengthening reached at the end of the preﬂex phase was analyzed for each perturbation level. Bars in (A,B) and (C,D) indicate the work done by skinned muscle ﬁbers and the isolated contractile element at dynamic-scenario and quasistatic-scenario, respectively. We calculated and indicate the area of the normalized works loops (Figure 3). Hence, there is no need to match the parameters of the hopping simulation to experimental muscle size. FIGURE 5 Inﬂuence of velocity adaptation on preﬂex work is represented. (A) shows the dissipated energies at dynamic-scenario experiments after the short- range stiffness till the end of the ‘step-up’ perturbation phase (shaded area shown in the inset). Elastic contribution is kept equal for all perturbation states. Thus, the difference in energy will be caused by the difference in velocity proﬁles. (B) shows the preﬂex work difference between the dynamic-scenario and quasistatic-scenario experiments for each perturbation level. The preﬂex work is shown in the insets as a shaded area and for multiple scenarios. Frontiers in Bioengineering and Biotechnology frontiersin.org 08 FIGURE 4 The amount of preﬂex work done by skinned ﬁbers and the isolated contractile element of the Hill-type muscle model for all perturbation states and activity levels. In the dynamic-scenario analysis, work done during the preﬂex phase was calculated as the area under the force curve. Shaded areas in the insets (A) and (B) indicate changing perturbation levels. In the quasistatic-scenario analysis, the area till the lengthening reached at the end of the preﬂex phase was analyzed for each perturbation level. 4 Discussion Bars in (A,B) and (C,D) indicate the work done by skinned muscle ﬁbers and the isolated contractile element at dynamic-scenario and quasistatic-scenario, respectively. We calculated and indicate the area of the normalized works loops (Figure 3). Hence, there is no need to match the parameters of the hopping simulation to experimental muscle size. FIGURE 4 The amount of preﬂex work done by skinned ﬁbers and the isolated contractile element of the Hill-type muscle model for all perturbation states and activity levels. In the dynamic-scenario analysis, work done during the preﬂex phase was calculated as the area under the force curve. Shaded areas in the insets (A) and (B) indicate changing perturbation levels. In the quasistatic-scenario analysis, the area till the lengthening reached at the end of the preﬂex phase was analyzed for each perturbation level. Bars in (A,B) and (C,D) indicate the work done by skinned muscle ﬁbers and the isolated contractile element at dynamic-scenario and quasistatic-scenario, respectively. We calculated and indicate the area of the normalized works loops (Figure 3). Hence, there is no need to match the parameters of the hopping simulation to experimental muscle size. FIGURE 5 Inﬂuence of velocity adaptation on preﬂex work is represented. (A) shows the dissipated energies at dynamic-scenario experiments after the short- range stiffness till the end of the ‘step-up’ perturbation phase (shaded area shown in the inset). Elastic contribution is kept equal for all perturbation states. Thus, the difference in energy will be caused by the difference in velocity proﬁles. (B) shows the preﬂex work difference between the dynamic-scenario and quasistatic-scenario experiments for each perturbation level. The preﬂex work is shown in the insets as a shaded area and for multiple scenarios. FIGURE 5 Inﬂuence of velocity adaptation on preﬂex work is represented. (A) shows the dissipated energies at dynamic-scenario experiments after the short- range stiffness till the end of the ‘step-up’ perturbation phase (shaded area shown in the inset). Elastic contribution is kept equal for all perturbation states. Thus, the difference in energy will be caused by the difference in velocity proﬁles. (B) shows the preﬂex work difference between the dynamic-scenario and quasistatic-scenario experiments for each perturbation level. The preﬂex work is shown in the insets as a shaded area and for multiple scenarios. 08 Frontiers in Bioengineering and Biotechnology frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 FIGURE 6 Short-range stiffness of muscle ﬁbers during the dynamic-scenario (A). 4 Discussion Stiffness amount during the short-range stiffness lengthening during quasistatic-scenario experiments (B) for all perturbation and activity levels are shown in the bar charts. Boundary conditions for the stiffness calculations for both speed conditions are shown as insets. In addition, velocity-length proﬁles during the preﬂex phase are presented in (C) and (D) for actual and quasistatic-scenario experiments, respectively. Thick lines show the short-range stiffness phase, and thin lines present the remaining part of the preﬂex phase. FIGURE 6 Short-range stiffness of muscle ﬁbers during the dynamic-scenario (A). Stiffness amount during the short-range stiffness lengthening during quasistatic-scenario experiments (B) for all perturbation and activity levels are shown in the bar charts. Boundary conditions for the stiffness calculations for both speed conditions are shown as insets. In addition, velocity-length proﬁles during the preﬂex phase are presented in (C) and (D) for actual and quasistatic-scenario experiments, respectively. Thick lines show the short-range stiffness phase, and thin lines present the remaining part of the preﬂex phase. of the stretch, until the end of the short-range stiffness phase at 0.59 ~lCE (Figure 3A). Our observation is in agreement with the reported constant short-range stiffness for stretch velocity ranges similar to ours (3 lopt/s to 5 lopt/s) (Rack and Westbury, 1974; Pinniger et al., 2006). mechanical properties of an individual muscle ﬁber result in the modulation of the force output to perturbation during locomotion. We extracted the boundary conditions from a hopping simulation for three levels of perturbations. Here, we discuss surprising outcomes that we observed from our in vitro experiments and simulations. As hypothesized, muscles modulate their force response to the stretch velocities. However, this modulation is not the main contributor to the preﬂex work. In addition, we observed that muscle’s intrinsic properties are tunable by changing the activity level. After the short-range stiffness phase, i.e., from 0.59 ~lCE to the end of preﬂex, the force response became nonlinear, and velocity adaptation occurred. In this phase, higher stretch velocities cause higher forces (Figure 3A) and preﬂex work (Figure 4A). Both observations are in agreement with the reported work increment associated with increasing stretch velocity for the eccentric phase of ramp-like stretch-shortening cycles (Tomalka et al., 2021). Frontiers in Bioengineering and Biotechnology frontiersin.org 4.2 Tuning the force and energy response by activity level Humans increase muscle activity in preparation for a step-down perturbation, as previously shown (Müller et al., 2015; Müller et al., 2012). The increased muscle activity strategy increases walking robustness (Haeuﬂe et al., 2018). To test whether increased activity leads to higher muscle stiffness and work in this scenario, we conducted muscle ﬁber experiments with activity levels of 5%, 15%, and 25% for each perturbation case while keeping the kinematics identical. The results conﬁrm previous ﬁndings (Campbell et al., 2014) that the short-range stiffness increases with activity level (Figure 6A). As short-range stiffness is likely due to the stretch of attached cross-bridges (Getz et al., 1998; Pinniger et al., 2006), an increase in short-range stiffness can be explained by the increasing number of attached cross-bridges with increasing activity level (Metzger and Moss, 1990). Interestingly, even though the dynamic behaviors during the preﬂex phase differ between quasistatic-scenario and dynamic- scenario conditions (Figures 3A, B), we observed almost equal amounts of mechanical work at quasistatic-scenario compared to dynamic-scenario stretches for each activity level (Figures 4A, B). Possibly, myosin heads are detached forcibly from actin at high velocities during eccentric contractions. This will decrease muscle force generation (Weidner et al., 2022) observed during in situ and in vitro experiments (Grifﬁths et al., 1980; Krylow and Sandercock, 1997; Till et al., 2008; Fukutani et al., 2019; Tomalka et al., 2020; Weidner et al., 2022). On the other hand, during the quasistatic- scenario stretches, ultra-slow-speed stretches allow cross-bridges to bind easier and longer than during rapid contractions (Huxley, 1957; Herzog, 2018). Hence, similar forces during the preﬂex stretch phase result in similar amounts of energy dissipation in quasistatic- scenario and dynamic-scenario experiments. Interestingly, even though the dynamic behaviors during the preﬂex phase differ between quasistatic-scenario and dynamic- scenario conditions (Figures 3A, B), we observed almost equal amounts of mechanical work at quasistatic-scenario compared to dynamic-scenario stretches for each activity level (Figures 4A, B). Additionally, we found that higher activity levels result in signiﬁcantly higher preﬂex work (Figure 4A). Tomalka et al. (2021) likewise reported an increase in work with an increasing number of active cross-bridges in the eccentric phase of the ramp- like stretch-shortening cycles. An increase in preﬂex work with increasing activity level might be explained by an increasing number of forcibly detached cross-bridges after the short-range stiffness phase. 4.1 Muscle response to perturbations at dynamic-scenario In previous isovelocity stretch experiments, the initial force response (i.e., short-range stiffness) was velocity dependent (Rack and Westbury, 1974; Pinniger et al., 2006), especially when the strain rate was varied over several orders of magnitude. For example, Pinniger et al. (2006) showed that the initial force response differs between slow (0.1 lopt/s) and fast (2 lopt/s) stretches. However, for contractions faster than 2 lopt/s they observed no signiﬁcant difference in the short-range stiffness. Weidner et al. (2022) observed differences in the short- range stiffness between 0.01 lopt/s and 1 lopt/s stretches. Our results align with these previous ﬁndings: while the short-range stiffness appears to be velocity-independent within the range of velocities tested in our dynamic-scenario experiments (reached peak velocities from 3 lopt/s to 5 lopt/s; Figure 6C), the comparison to the quasistatic- scenario experiments (reached maximum peak velocity of 0.05 lopt/s, Figure 6D) revealed that the short-range stiffness actually reﬂects a muscle behavior that varies with large differences of velocity. between the perturbation cases become signiﬁcant (Figure 4A). We ﬁnd a rising trend in dissipated energy with the increase of stretch velocity from step-up to step-down perturbations. Hence, muscles adjust their work response primarily due to a change of stretch caused by locomotion perturbation. 4.2 Tuning the force and energy response by activity level The number of forcibly detached cross-bridges might increase (at the given stretch kinematics) as a fraction of the increasing number of attached cross-bridges with activity level (Wahr and Rall, 1997). Forced detachment of cross-bridges is expected (Weidner et al., 2022) in the range of the tested stretch velocities (3 lopt/s to 5 lopt/s; Figure 6). Additionally, viscoelastic properties of non-cross-bridge structures (e.g., titin) might contribute to energy dissipation in a velocity-dependent manner (Herzog et al., 2014; Freundt and Linke, 2019; Tomalka et al., 2021). Furthermore, with higher activity, the differences between perturbation cases become more prominent, and, for 25%, even signiﬁcant (Figure 4A). Possibly, myosin heads are detached forcibly from actin at high velocities during eccentric contractions. This will decrease muscle force generation (Weidner et al., 2022) observed during in situ and in vitro experiments (Grifﬁths et al., 1980; Krylow and Sandercock, 1997; Till et al., 2008; Fukutani et al., 2019; Tomalka et al., 2020; Weidner et al., 2022). On the other hand, during the quasistatic- scenario stretches, ultra-slow-speed stretches allow cross-bridges to bind easier and longer than during rapid contractions (Huxley, 1957; Herzog, 2018). Hence, similar forces during the preﬂex stretch phase result in similar amounts of energy dissipation in quasistatic- scenario and dynamic-scenario experiments. 4.4 Muscle ﬁbers versus Hill-type muscle model Since Hill’s empirical investigations of muscle contraction dynamics, Hill-type models have played a crucial role in biomechanics research (Hill, 1938; Rode and Siebert, 2017). These models have been improved over the years, but they are still limited in predicting muscle forces, especially during eccentric (lengthening) contractions (Till et al., 2008; Siebert et al., 2021). Surprisingly, our results show that the magnitude and trends in mechanical work predicted by the Hill-type contractile element model are similar to the work of muscle ﬁbers for fast eccentric contractions (Figures 4A, C). This is an unexpected outcome since the force response of the Hill-type muscle model and muscle ﬁbers differs (Figures 3A, C). We show that the main source of preﬂex-work modulation to perturbation height is the amount of muscle stretch rather than the viscous force adaptation. Consequently, humans and animals may tune their muscle stiffness during the short-range stiffness phase (Figure 6A) and their muscle work after the short-range stiffness phase (Figure 5A) utilizing the activity level. Thus, increased muscle stiffness and work in preparation for an expected perturbation are possible by increasing the muscle pre-activity level. 4.1 Muscle response to perturbations at dynamic-scenario Two factors contribute to the increasing preﬂex work in our study. The ﬁrst factor is the higher force when assuming the same stretch is considered for calculating the preﬂex work (Figure 5A, inset). For the speciﬁed area, all energetic differences between perturbations are the result of muscle ﬁbers’ viscous behavior since stretch amounts are identical. In that case, we observed no signiﬁcant differences in preﬂex work between perturbations. Second, higher velocities cause a larger ﬁbers stretch in the preﬂex phase (Figure 3A). If the larger stretch is fully considered, differences in preﬂex work During dynamic-scenario experiments, muscle ﬁbers initially react elastically to the sudden perturbation, known as short-range stiffness (Rack and Westbury, 1974; Kirsch et al., 1994). Muscles then transition into a viscoelastic behavior (Figure 3A). In the short- range stiffness region, we found no signiﬁcant changes in ﬁber work, despite of different perturbations changed stretch velocities (Figure 6C). We calculated ﬁber work starting at the beginning 09 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 Comparison of dynamic-scenario and quasistatic-scenario experiments show that velocity is not only adding a viscous behavior to the response (Figure 3A, B), but also a visible short- range stiffness contribution (Figure 6). In previous isovelocity stretch experiments, the initial force response (i.e., short-range stiffness) was velocity dependent (Rack and Westbury, 1974; Pinniger et al., 2006), especially when the strain rate was varied over several orders of magnitude. For example, Pinniger et al. (2006) showed that the initial force response differs between slow (0.1 lopt/s) and fast (2 lopt/s) stretches. However, for contractions faster than 2 lopt/s they observed no signiﬁcant difference in the short-range stiffness. Weidner et al. (2022) observed differences in the short- range stiffness between 0.01 lopt/s and 1 lopt/s stretches. Our results align with these previous ﬁndings: while the short-range stiffness appears to be velocity-independent within the range of velocities tested in our dynamic-scenario experiments (reached peak velocities from 3 lopt/s to 5 lopt/s; Figure 6C), the comparison to the quasistatic- scenario experiments (reached maximum peak velocity of 0.05 lopt/s, Figure 6D) revealed that the short-range stiffness actually reﬂects a muscle behavior that varies with large differences of velocity. Comparison of dynamic-scenario and quasistatic-scenario experiments show that velocity is not only adding a viscous behavior to the response (Figure 3A, B), but also a visible short- range stiffness contribution (Figure 6). Frontiers in Bioengineering and Biotechnology frontiersin.org 4.3 Dynamic- versus quasistatic-scenario During the preﬂex phase of the quasistatic-scenario experiments, when muscles are stretched with negligible velocities, they respond with a linear force increase which is almost the same regardless of the perturbation case (Figure 3B). Our quasistatic-scenario experiments and simulations proved that the force-length relation of the Hill-type muscle model could 10 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 10.3389/fbioe.2023.1150170 1978), which on the one hand is speed-dependent and on the other hand occurs directly after the end of the short-range stiffness (Weidner et al., 2022). The force and work responses show that the modeling of eccentric muscle behavior needs to be modiﬁed for better estimation of the dynamic response to perturbations during fast eccentric contractions. In addition, the effect of the neural reﬂex response after the preﬂex phase will have a signiﬁcant effect on the post-preﬂex force generation (Nichols and Houk, 1976) and should also be examined in further experiments with an appropriately updated experimental setup. accurately estimate the length-dependent force and mechanical work response of muscle ﬁbers for the investigated conditions (relatively short muscle ﬁbers at low activity levels). Because the length-dependent behavior of muscle ﬁbers is the main force contributor during preﬂex, the Hill-type muscle model predicts work for the larger stretch in response to the fast contraction reasonably well. Although the Hill-type muscle model estimates work during preﬂex with good accuracy, it still requires improvements for better force prediction during fast contractions (Figures 3A, C). The short-range stiffness had previously been observed in other ﬁber experiments (Rack and Westbury, 1974; Tomalka et al., 2021; Weidner et al., 2022). We observed that the short-range stiffness was activity- and velocity-dependent (Figure 6), at least for the velocity difference between dynamic-scenario and quasistatic- scenario experiments (Figures 3A, B). However, our Hill-type muscle model cannot generate the high-stiffness response of a short-range stiffness, since short-range stiffness is not a built-in mechanical property (Haeuﬂe et al., 2014). So far, the short-range stiffness model proposed by Cui et al. (2008) has been implemented in several musculoskeletal simulations to study the inﬂuence of short-range stiffness on the end point stiffness of the human arm in static postures (Hu et al., 2011) and postural stability while standing (De Groote et al., 2017). 4.5 Study limitations This study aimed at analyzing how a single muscle ﬁber reacts to ground perturbations in real life. It is known that the temperature inﬂuences the muscle properties (e.g., Fmax, Vmax, (Ranatunga, 1984; Stephenson and Williams, 1985; Zhao and Kawai, 1994)) and thus the force response to disturbances. Therefore, temperature inﬂuences on the force response to perturbations should be investigated in future studies. We conducted single-leg hopping simulations using a Hill-type muscle model as a knee extensor muscle to generate kinematic boundary conditions for in vitro experiments. However, Hill-type muscle models have limitations discussed in previous chapters, and simulation and real-life muscle lengthening may differ. Additionally, our in vitro experimental setup allows only constant activity levels. Even though in the hopping simulations after the preﬂex phase activity rises, due to the setup limitations, we perform the kinematic analysis with the constant preﬂex activity level, which is not the case in in vivo hopping (Moritz and Farley, 2004) and locomotion (Müller et al., 2015). Thus, we only focused our analysis on the preﬂex phase. We assume a preﬂex time-span of 30 ms based on the reﬂex delay scaling found by More et al. (2010). Since we do not consider a full work loop, this study’s design does not directly allow us to calculate damping, i.e., the amount of energy dissipation in a full cycle. However, the velocity- dependent modulation of preﬂex work indicates a viscous-like response, which we identify as a damping behavior. The force modulation to the perturbation velocity after the short-range stiffness (Figures 3A, C) is also not accurately modeled in the Hill-type muscle model. Here, the Hill-type muscle model operates in the plateau region of the eccentric force-velocity relation (Figure 2). The model, therefore, does not show any modulation of the force due to the perturbation-related changes in ﬁber velocity, in contrast to the observations in the experiments (Figures 3A, C). This plateau-form of the eccentric force-length relation was introduced by van Soest and Bobbert (1993), while their results are consistent with our simulation data, they do not explain the experimental results of the present study. Possible reasons for the deviation of the experiment can be the starting length of the contraction, the sub-maximal activity level or the underlying model. However, the results of Krylow and Sandercock (1997) suggest that the starting length has no inﬂuence on the point of occurrence of the eccentric force- velocity-relation’s plateau. 4.3 Dynamic- versus quasistatic-scenario These simulation studies showed that including short-range stiffness in a Hill-type muscle model improves estimation of joint angles, torques and stiffness (Hu et al., 2011), as well as postural stability against external perturbations (De Groote et al., 2017). Together with our results, we expect that Hill-type muscle models that feature short-range stiffness should provide a better force estimation at and immediately after impact. Therefore, we consider short-range stiffness an essential model feature for the understanding of gait mechanics leading to stable locomotion. 4.5 Study limitations Regarding the sub-maximal activity level and its effects, it is known that the calcium concentration has an inﬂuence on the cross-bridge kinetics (Brenner, 1988). Nevertheless, to the best of our knowledge, there is no study that looked at the dependency of the eccentric force-velocity relation on the activity level within the contractile element only. A likely explanation for the discrepancy is simpliﬁcation of the contraction by the model. For example, the Hill-type muscle model lacks the implementation of “Give” (Flitney and Hirst, Frontiers in Bioengineering and Biotechnology References De Groote, F., Allen, J. L., and Ting, L. H. (2017). Contribution of muscle short-range stiffness to initial changes in joint kinetics and kinematics during perturbations to standing balance: A simulation study. J. Biomechanics 55, 71–77. doi:10.1016/j.jbiomech.2017.02.008 Bayer, A., Schmitt, S., Günther, M., and Haeuﬂe, D. (2017). The inﬂuence of biophysical muscle properties on simulating fast human arm movements. Comput. methods biomechanics Biomed. Eng. 20, 803–821. doi:10.1080/10255842.2017.1293663 Blickhan, R., Seyfarth, A., Geyer, H., Grimmer, S., Wagner, H., and Günther, M. (2007). Intelligence by mechanics. Philosophical Trans. R. Soc. A Math. Phys. Eng. Sci. 365, 199–220. doi:10.1098/rsta.2006.1911 Blickhan, R., Seyfarth, A., Geyer, H., Grimmer, S., Wagner, H., and Günther, M. (2007). Intelligence by mechanics. Philosophical Trans. R. Soc. A Math. Phys. Eng. Sci. 365, 199–220. doi:10.1098/rsta.2006.1911 Dick, T. J. M., Biewener, A. A., and Wakeling, J. M. (2017). Comparison of human gastrocnemius forces predicted by Hill-type muscle models and estimated from ultrasound images. J. Exp. Biol. 154807, 1643–1653. jeb. doi:10.1242/jeb.154807 Flitney, F., and Hirst, D. (1978). Cross-bridge detachment and sarcomere’give’during stretch of active frog’s muscle. J. Physiology 276, 449–465. doi:10.1113/jphysiol.1978. sp012246 Brenner, B. (1988). 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Force- and moment-generating capacities of muscles in the distal forelimb of the horse. J. Anat. 203, 101–113. doi:10.1046/j.1469-7580.2003.00206.x Gerritsen, K. G., van den Bogert, A. J., Hulliger, M., and Zernicke, R. F. (1998). Intrinsic muscle properties facilitate locomotor control—A computer simulation study. Mot. control 2, 206–220. doi:10.1123/mcj.2.3.206 Campbell, K. S., Jitandrakumar, R., and Moss, R. L. (2014). Cycling cross-bridges increase myocardial stiffness at submaximal levels of Ca2+ activation. Biophysical J. 84, 3807–3815. doi:10.1016/S0006-3495(03)75108-X Getz, E. B., Cooke, R., and Lehman, S. L. (1998). Data availability statement Max Planck Research School for Intelligent Systems (IMPRS-IS) for supporting FI. We acknowledge support by Open Access Publishing Fund of University of Tübingen. All experimental data and the Matlab/Simulink ﬁles needed to generate manuscript results and ﬁgures are available in Zenodo (doi. org/10.5281/zenodo.7774608). Author contributions 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. MA, SW, AB-S, TS, and DH conceptualized the project. FI and MA conducted computer simulations. MA and SW conducted the experiments and analyzed the data. MA and SW prepared the manuscript, and all authors revised and approved the submitted version. Ethics statement The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. The experiment was conducted according to the guidelines of ARRIVE and approved according to the German animal protection law (Tierschutzgesetz §4(3), permit no. T170/18ST). 5 Conclusion Previous experimental and simulation studies indicated that muscles’ preﬂex capability to adjust force to unexpected ground conditions is essential in stabilizing hopping and locomotion. Our study conﬁrms these ﬁndings and shows three mechanisms: (1) muscle force adapts to the change in stretch velocity caused by a perturbation; (2) the overall ﬁber stretch in the preﬂex duration is larger for larger stretch velocities resulting in increased preﬂex work; (3) with increasing muscle activity short-range stiffness and muscle force increase. Mechanism (1) is the hypothesized viscous effect of the force- velocity relationship but plays a minor role compared to the mechanism (2). Together, (1) and (2) result in a beneﬁcial and signiﬁcant modulation of muscle force to perturbations and thus conﬁrm the preﬂex hypothesis. Mechanism (3) allows for a simple neuronal strategy to tune the muscle properties to ground conditions and unexpected perturbations and aligns with feed-forward strategies observed in human locomotion (Müller et al., 2012). 11 frontiersin.org Araz et al. 10.3389/fbioe.2023.1150170 10.3389/fbioe.2023.1150170 Funding The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fbioe.2023.1150170/ full#supplementary-material This work was funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation)—449912641-HA 7170/3, SI 841/22-1 and BA 7275/3-1. The authors thank the International References Muscle prestimulation tunes velocity preﬂex in simulated perturbed hopping. Sci. Rep. 13, 4559. doi:10.1038/s41598-023-31179-6 Rode, C., and Siebert, T. (2017). “Muscle-like actuation for locomotion,” in Bioinspired legged locomotion. Editors A. S. Maziar, and A. 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https://openalex.org/W2901629839	https://europepmc.org/articles/pmc6251111?pdf=render	English	null	Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing	Virology journal	2,018	cc-by	11,094	Open Access Rapid virulence prediction and identification of Newcastle disease virus genotypes using third-generation sequencing Salman L. Butt1,2, Tonya L. Taylor1, Jeremy D. Volkening3, Kiril M. Dimitrov1, Dawn Williams-Coplin1, Kevin K. Lahmers4, Patti J. Miller1,6, Asif M. Rana5, David L. Suarez1, Claudio L. Afonso1* and James B. Stanton2* Butt et al. Virology Journal (2018) 15:179 https://doi.org/10.1186/s12985-018-1077-5 Butt et al. Virology Journal (2018) 15:179 https://doi.org/10.1186/s12985-018-1077-5 * Correspondence: Claudio.Afonso@ars.usda.gov; jbs@uga.edu * Correspondence: Claudio.Afonso@ars.usda.gov; jbs@uga.edu * Correspondence: Claudio.Afonso@ars.usda.gov; jbs@uga.edu p g j g 1Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA 30605, USA 1Southeast Poultry Research Laboratory, US National Poultry Research Center, Agricultural Research Service, USDA, 934 College Station Road, Athens, GA 30605, USA 2 2Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA Full list of author information is available at the end of the article 2Department of Pathology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA Full list of author information is available at the end of the article Abstract Background: Newcastle disease (ND) outbreaks are global challenges to the poultry industry. Effective management requires rapid identification and virulence prediction of the circulating Newcastle disease viruses (NDV), the causative agent of ND. However, these diagnostics are hindered by the genetic diversity and rapid evolution of NDVs. Methods: An amplicon sequencing (AmpSeq) workflow for virulence and genotype prediction of NDV samples using a third-generation, real-time DNA sequencing platform is described here. 1D MinION sequencing of barcoded NDV amplicons was performed using 33 egg-grown isolates, (15 NDV genotypes), and 15 clinical swab samples collected from field outbreaks. Assembly-based data analysis was performed in a customized, Galaxy-based AmpSeq workflow. MinION-based results were compared to previously published sequences and to sequences obtained using a previously published Illumina MiSeq workflow. Results: For all egg-grown isolates, NDV was detected and virulence and genotype were accurately predicted. For clinical samples, NDV was detected in ten of eleven NDV samples. Six of the clinical samples contained two mixed genotypes as determined by MiSeq, of which the MinION method detected both genotypes in four samples. Additionally, testing a dilution series of one NDV isolate resulted in NDV detection in a dilution as low as 101 50% egg infectious dose per milliliter. This was accomplished in as little as 7 min of sequencing time, with a 98.37% sequence identity compared to the expected consensus obtained by MiSeq. Conclusion: The depth of sequencing, fast sequencing capabilities, accuracy of the consensus sequences, and the low cost of multiplexing allowed for effective virulence prediction and genotype identification of NDVs currently circulating worldwide. The sensitivity of this protocol was preliminary tested using only one genotype. After more extensive evaluation of the sensitivity and specificity, this protocol will likely be applicable to the detection and characterization of NDV. words: Newcastle disease virus, RNA, Genotype, Nanopore sequencing, Rapid sequencing, MinION, N Background Virulence of NDV is best assayed through in vivo pathogenicity studies [5], but due to the cost and time constraints associated with such methods, reverse transcriptase-quantitative PCR (RT-qPCR) and sequencing of the F gene cleavage site are used to predict NDV virulence [6, 7]. Genotyping of NDV is commonly achieved through sequencing of the coding sequence of the fusion gene [8], which also allows for prediction of virulence. Preliminary genotyping can be accomplished through partial fusion gene sequencing (i.e., variable region) [9]. For detection of NDV, PCR assays avoid the highly variable fusion gene and instead target more conservative regions of the genome (i.e., matrix and polymerase genes) [10–14]; however, while this increases the applicability of these assays across genotypes, these as- says lack applicability for virulence and genotypic deter- mination. For example, while fusion-based assays can be used for detection [10, 15], the variability of this region, which makes it useful for genotyping, hinders the univer- sal applicability of any single primer set [11, 12] and often requires screening samples with a different PCR assay prior to pathotyping [15]. Furthermore, for most current methods, detection, genotyping, and virulence prediction rely on Sanger sequencing; thus, they lack multiplexing capability and have limited sequencing depth, which com- plicates detection of mixed infections. In summary, there is a need to develop a method that will sensitively and rapidly detect NDV from multiple genotypes, while also providing genotype and virulence predictions. As with any deep sequencing platform, the sequence analysis approach is integral for accurate interpretation. Primarily, two approaches for taxonomic profiling of mi- crobial sequencing data have been employed: read-based and de novo assembly-based classifications. Read-based metagenomic classification software has been used for identification of microbial species from high-throughput sequencing data [23, 31–33]. Although the sequencing ac- curacy of the MinION is improving, the raw single-read error rate of nearly 10% [34] may limit the accuracy of this approach for Nanopore data [31], especially when attempt- ing to subspecies level differentiation. De novo approaches that use quality-based filtering and clustering of reads [35], or use consensus-based error correction of Nanopore se- quencing reads have been reported [36]; however, these are not optimized for amplicon sequencing data. In this study, a specific and rapid protocol, using the MinION sequencer, was developed to detect represen- tative isolates from all currently circulating (excluding the Madagascar-limited genotype XI) genotypes of NDV. Background sequencing [19], transcriptomics, and metagenomics [20, 21]. However, high capital investments and relatively long turnaround times limit the widespread use of these next-generation sequencing (NGS) platforms, especially in developing countries [22]. Recent improvements in third-generation sequencing, including those introduced by Oxford Nanopore Technologies (ONT) [23], increase the utility of high-throughput sequencing as a useful tool for surveillance and pathogen characterization [24]. Among the transformative advantages of ONT’s sequen- cing technology are the ability to perform real-time se- quence analysis with a short turnaround time [25], the portability of the MinION device, the low startup cost compared to other high-throughput platforms, and the ability to sequence up to several thousand bases from individual RNA or DNA molecules. The MinION de- vice has been successfully used to evaluate antibiotic resistance genes from several bacterial species [26, 27], obtain complete viral genome sequences of an influenza virus [28] and Ebola virus [29], and detect partial viral genome sequences (e.g., Zika virus [30] and poxviruses [25]) by sequencing PCR amplicons (AmpSeq). The MinION, therefore, represents an opportunity to take infectious disease diagnostics a step further and to per- form rapid identification and genetic characterization of infectious agents at a lower cost. g Newcastle disease (ND) is one of the most important infectious diseases of poultry and is a major economic burden to the global poultry industry. Virulent strains of avian paramyxovirus 1 (APMV-1), commonly known as Newcastle disease virus (NDV) [1], are the cause of ND and have been recently reclassified as avian avulavirus-1 (AAvV-1) [2]. Newcastle disease viruses are a highly diverse group of viruses with two distinct classes, 19 accepted genotypes and a wide host range including domestic and wild bird species. In addition to the genotypic diversity of NDVs, these viruses are also diverse in their virulence. This includes low virulent vi- ruses, whose replication is limited to the respiratory and digestive tracts and typically cause clinically in- apparent infections, to highly virulent viruses that cause acute disease with high mortality rates [1, 3]. The global spread, constant evolution, varying virulence, and the wide host range of NDV are challenges to the control of ND [4]. Effective control of ND is dependent on specific diag- nostic testing, which is typically oriented towards detec- tion, genotyping, or prediction of virulence. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Butt et al. Virology Journal (2018) 15:179 Butt et al. Virology Journal (2018) 15:179 Page 2 of 14 Page 2 of 14 Background This protocol was also tested on 15 clinical swab samples collected from chickens during disease outbreaks. Additionally, a Galaxy-based, de novo AmpSeq workflow is presented that results in accurate final consensus sequences allowing for accurate genotype and virulence prediction. This study represents the first step towards developing AmpSeq as a diagnostic tool for NDV. Rapid advances in nucleic acid sequencing have led to different sequencing platforms [16, 17] being widely ap- plied for identification of novel viruses [18], whole genome Butt et al. Virology Journal (2018) 15:179 Butt et al. Virology Journal (2018) 15:179 Page 3 of 14 Page 3 of 14 Page 3 of 14 Viruses and clinical samples Thirty-three NDV isolates, representing 15 different genotypes of different virulence, and ten other avian avulaviruses (AAvV 2–10 and AAvV-13) from the Southeast Poultry Research Laboratory (SEPRL) reposi- tory, were propagated in 9–11-day-old specific patho- gen free (SPF) eggs [37] and the harvested allantoic fluids were used in this study. Additionally, 15 oral and cloacal swab samples collected from chickens during disease outbreaks in Pakistan in 2015 were collected, and the resulting swab fluid was shipped on dry ice, and then stored at −80 °C. RNA was extracted as described below for both egg-grown and clinical swab samples. The background information of the egg-grown isolates and the clinical samples is summarized in Additional file 1: Table S1 and Table S2, respectively. Comparison of AmpSeq protocol to RT-qPCR assay p p q p q y For comparison of this MinION-based protocol with the matrix gene reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) assay [10], both methods were used on a dilution series from a single isolate. NDV (LaSota strain) from the SEPRL repository was cultured in SPF 9–11-days-old eggs and the harvested allantoic fluids were diluted to titers ranging from 106 to 101 EID50/mL in brain-heart infusion broth. RNA was extracted from dilu- tions, and DNA libraries were prepared following the same protocols as described above. Amplicons from each of the dilutions were barcoded separately. At the pooling step, equal concentrations of barcoded amplicons from different dilutions of LaSota were pooled together in sin- gle tube. Dilutions, extractions, library construction, and sequencing were performed twice (run 1 and run 2). RNA extraction Total RNA from each sample was extracted from infec- tious allantoic fluids or directly from clinical swab media using TRIzol LS (Thermo Fisher Scientific, USA) follow- ing the manufacturer’s instructions. RNA concentrations were determined by using Qubit® RNA HS Assay Kit on a Qubit® fluorometer 3.0 (Thermo Fisher Scientific, USA). Methods (Oxford Nanopore Technologies, UK) in conjunction with the Ligation Sequencing Kit 1D (SQK-LSK108) [23] as per manufacturer’s instructions. Briefly, each of the amplicons were diluted to 0.5 nM for barcoding and amplified using LongAmp Taq 2X Master Mix (New England Biolabs, USA) with the following conditions; 95 °C for 3 min; 15 cycles of 95 °C for 15 s; 62 °C for 15 s, 65 °C for 50 s, followed by 65 °C for 50 s. The bar- coded amplicons were bead purified, pooled into a sin- gle tube, end prepped, dA tailed, bead purified, and ligated to the sequencing adapters per manufacturer’s instructions. Final DNA libraries were bead purified and stored frozen until used for sequencing. Viruses and clinical samples Amplicon synthesis and MinION library preparation Amplicon synthesis and MinION library preparation Approximately 20 ng (in 5 μl) of RNA was reverse tran- scribed, and cDNA was amplified with target-specific primers using the SuperScript™III One-Step RT-PCR System (Thermo Fisher Scientific, USA). Previously published primers (4331F and 5090R) [9, 38] were used in this protocol to target NDV; however, the primers were tailed with universal adapter sequence of 22 nu- cleotides (in bold font) to allow PCR-based barcoding: 4331F Tailed: 5′-TTTCTGTTGGTGCTGATATTGCG AGGTTACCTCYACYAAGCTRGAGA-3′; 5090R Tailed: 5′-ACTTGCCTGTCGCTCTATCTTCTCATTAACAAA YTGCTGCATCTTCCCWAC-3′). The thermocycler con- ditions for the reaction were as follows: 50 °C for 30 min; 94 °C for 2 min; 40 cycles of 94 °C for 15 s, 56 °C for 30 s, and 68 °C for 60 s, followed by 68 °C for 5 min. The reac- tion amplified a 788 base pair (bp) NDV product (832 bp including primer tails) for all genotypes, which included 173 bp of the 3′ region of the end of the M gene and 615 bp of the 5′ end of the F gene (sizes and primer loca- tions based on the Genotype V strain). Amplified DNA was purified by Agencourt AMPure XP beads (Beckman Coulter, USA) at 1.6:1 volumetric bead-to-DNA ratio and quantified using the dsDNA High Sensitivity Assay kit on a Qubit® fluorometer 3.0. MinION-compatible DNA li- braries were prepared with approximately 1 μg of bar- coded DNA in a total volume of 45 μL using nuclease-free water and using the 1D PCR Barcoding Amplicon Kit The same extracted RNA was also used as the input into the RT-qPCR using the AgPath-ID one-step RT-PCR Kit (Ambion, USA) on the ABI 7500 Fast Real-Time PCR sys- tem following the previously described protocols [10]. Sequencing by MinION q g y The libraries were sequenced with the MinION Nano- pore sequencer [23]. A new FLO-MIN106 R9.4 flow cell, stored at 4 °C prior to use, was allowed to equili- brate to room temperature for 10 min before priming it for sequencing. The flow cell was primed with running buffer as per manufacturer’s instructions. The pooled DNA libraries were prepared by combining 12 μL of the libraries with 2.5 μL nuclease-free water, 35 μL RBF, and 25.5 μL library loading beads. After the MinION Platform QC run, the DNA library was loaded into the MinION flow cell via the SpotON port. The standard 48-h 1D sequencing protocol was initiated using the MinKNOW software v.5.12. Detailed information for all MinION runs in this study is provided in Additional file 1: Table S3. The complete steps from RNA isolation to MinION se- quencing were performed twice for egg-grown viruses. One run consisted of six egg-grown isolates from different Butt et al. Virology Journal (2018) 15:179 Butt et al. Virology Journal (2018) 15:179 Page 4 of 14 Page 4 of 14 Butt et al. Virology Journal (2018) 15:179 based on the summary table produced by Albacore. Pore- chop v0.2.2 [40] was used to demultiplex reads for each of the barcodes and trim the adapters at the ends of the reads by using default settings. Short reads (cutoff = 600 bp) were filtered out and the remaining reads were used as input to the in-house LAclust v0.002. LAclust per- forms single-linkage clustering of noisy reads based on alignment identity and length cutoffs from DALIGNER pairwise alignments [41] (minimum alignment coverage = 0.90, maximum identity difference = 0.35; minimum num- ber of reads to save cluster = 5; maximum reads saved per cluster = 200, minimum read length = 600 bp; rank mode = number of intracluster linkages; randomized input read order = yes). Read clusters generated by LAclust were then aligned using the in-house Amplicon aligner v0.001 to generate a consensus sequence. This tool optionally sub- samples reads (target depth used = 100), re-orients them as necessary, aligns them using Multiple Alignment using Fast Fourier Transform (MAFFT) [42] with highly relaxed gap opening and extension penalties, and calls a major- ity consensus. Next, each consensus was used as a ref- erence sequence for mapping the full unfiltered read clusters from LAclust with BWA-MEM and ONT2D settings [43, 44]. Sequencing by MinION The final consensus sequence for each genotypes representative of vaccine and virulent NDV strains (run 3: 6-sample pool). The other run consisted of these same six viruses and an additional 27 egg-grown NDV isolates (run 4: 33-sample pool). The clinical sam- ples (n = 15) were processed in runs 5, 6, and 7. A variable number of samples were pooled in these three sequencing runs to cluster libraries with similar concentrations. To determine the accuracy of consensus sequences at different sequencing time points for accurate identifica- tion of the NDV genotypes, the raw data (FAST5 files) obtained from the 10-fold serial dilution experiment (see above) were analyzed in subgroups based on time of acquisition and processed through the AmpSeq work- flow as described below. Sequencing by MiSeq For comparison between nucleotide sequences obtained from MinION and MiSeq (a high accuracy sequencing platform), 24 NDV isolates from the SEPRL repository that were used for MinION sequencing (representing each currently circulating genotype except XI and multiple sub-genotypes of NDV) and 15 clinical swab samples (al- lantoic fluid of cultured swab samples) were processed for target-independent NGS sequencing. Briefly, paired-end random sequencing was conducted from cDNA librar- ies prepared from total RNA using KAPA Stranded RNA-Seq kit (KAPA Biosystems, USA) as per manufac- turer’s instructions and as previously described [46]. All libraries for NGS were loaded into the 300-cycle MiSeq Reagent Kit v2 (Illumina, USA) and pair-end sequen- cing (2 × 150 bp) was performed on the Illumina MiSeq instrument (Illumina, USA). Pre-processing and de-novo assembly of the raw sequencing data was completed within the Galaxy platform using a previously described approach [19]. Time for data acquisition and analysis To determine the minimal sequencing time needed for acquisition of accurate full-length amplicon consensus sequences at different serial dilutions, 28,000 reads, which were obtained within the first 19 min of sequen- cing in the first serial dilution experiment (run 1), were analyzed. For all concentrations, the first read that aligned to the reference LaSota sequence was obtained within 5 min after the sequencing run started. To ob- tain consensus sequences (5 reads required to build a consensus sequence) only 5 min of sequencing time were required for concentrations 106–103 EID50/ml, which resulted in 99.18–100% sequence identity to the reference LaSota strain. Seven minutes were required to obtain NDV consensus sequences for the two lower concentrations: 101 EID50/ml = 8 reads, 98.77% identity and 102 EID50/ml = 5 reads, 98.37% identity (Table 2). After as little as 10 min of sequencing, the identity to the reference sequence was above 99% for even in the most dilute sample. Development of MinION data analysis workflow To analyze the Nanopore sequencing data, a custom, assembly-based AmpSeq workflow within the Galaxy plat- form interface [39] was developed, as diagrammed in Fig. 1. The MinION raw reads in FAST5 format were ar- chived (tar format) and uploaded into Galaxy workflow. The reads were base-called using the Albacore v2.02 (ONT). The NanoporeQC tool v0.001 (available in the Galaxy testing toolshed) was used to visualize read quality Fig. 1 Schematic diagram of customized Galaxy workflow for MinION sequence data analysis. Blue shading indicates pre-processing steps. Green shading indicates post-processing steps; assembly/output is shaded purple. Purple arrows indicate different inputs for final consensus calculation Fig. 1 Schematic diagram of customized Galaxy workflow for MinION sequence data analysis. Blue shading indicates pre-processing steps. Green shading indicates post-processing steps; assembly/output is shaded purple. Purple arrows indicate different inputs for final consensus calculation Butt et al. Virology Journal (2018) 15:179 Page 5 of 14 sample was refined by using Nanopolish v0.8.5 [45], which calculates an improved consensus using the read alignments and raw signal information from the original FAST5 files. After manually trimming primer sequences from both 3′ (25 bp) and 5′ (29 bp) ends, the obtained consensus sequences (734 bp) were BLAST searched against NDV customized database, which consisted NCBI’s nucleotide (nt) database and internal unpublished NDV sequences (NCBI database updated on May 23, 2018). deep-sequencing methods (MinION and MiSeq) de- scribed above. Pairwise nucleotide comparison between MinION and MiSeq was conducted using the Max- imum Composite Likelihood model [50]. The variation rate among sites was modeled with a gamma distribu- tion (shape parameter = 1). The analysis involved 54 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 691 positions in the final dataset. The evolutionary distances were inferred by pairwise analysis using the MEGA6 [48]. sample was refined by using Nanopolish v0.8.5 [45], which calculates an improved consensus using the read alignments and raw signal information from the original FAST5 files. After manually trimming primer sequences from both 3′ (25 bp) and 5′ (29 bp) ends, the obtained consensus sequences (734 bp) were BLAST searched against NDV customized database, which consisted NCBI’s nucleotide (nt) database and internal unpublished NDV sequences (NCBI database updated on May 23, 2018). Results Comparison to the matrix gene RT-qPCR assay Comparison to the matrix gene RT-qPCR assay Six, sequential, 10-fold dilutions (from 106 EID50/ml to 101 EID50/ml) from one NDV isolate (LaSota) were used to compare the ability of AmpSeq and RT-qPCR to detect low quantities of NDV. In each of the six dilutions, Amp- Seq and the matrix RT-qPCR detected NDV in all dilu- tions. AmpSeq resulted in 99.04–100.0% sequence identity to the LaSota isolate across all six dilutions in the first ex- periment (run 1) and 99.86–100.0% identity in the second experiment (run 2) (Table 1). Phylogenetic analysis The assembled consensus sequences from different NDV genotypes and sub-genotypes (6 sequences from MinION run 3, 33 sequences from run 4 and 24 sequences from MiSeq; a total of 62 sequences) and selected (minimum of one sequence from each genotype/subgenotype) se- quences from GenBank (n = 66) were aligned using Clus- talW [47] in MEGA6 [48]. Determination of the best-fit substitution model was performed using MEGA6, and the goodness-of-fit for each model was measured by corrected Akaike information criterion (AICc) and Bayesian infor- mation criterion (BIC) [48]. The final tree was constructed using the maximum-likelihood method based on the Gen- eral Time Reversible model as implemented in MEGA6, with 500 bootstrap replicates [49]. The available GenBank accession number for each sequence in the phylogenetic tree is followed by the, host name, country of isolation, strain designation, and year of isolation. PCR specificity and range of reactivity for NDV genotypes To determine the utility of the primers for the currently cir- culating NDV genotypes and the potential cross-reactivity for other AAvVs, which are relatively nonpathogenic in poultry but can confound diagnosis of NDV [50], total RNA from 43 AAvVs, including 23 AAvV-1 genotypes and sub-genotypes (15 different NDV genotypes, 8 different subgenotypes), as well as AAvV-2–10 and −13 (n = 10) Comparison of MinION and MiSeq sequence accuracy To assess the accuracy of the MinION AmpSeq con- sensus sequences, 24 samples were sequenced by both Butt et al. Phylogenetic analysis Virology Journal (2018) 15:179 Page 6 of 14 Table 1 Comparison of MinION sequencing to RT-qPCR for detection of NDV LaSota (runs 1 and 2) Dilution (EID50/ml) Total readsa Total NDV readsb Reads per consensusc Percent identityd Consensus length RT-qPCRe (Ct) R1f \| R2g R1 \| R2 R1 \| R2 R1 \| R2 R1 \| R2 R1 \| R2 10^6 6667 \| 11,366 6577 \| 10,861 200 \| 200 100 \| 100 734 \| 734 21.8, 21.1 \| 22.7, 22.7 10^5 4519 \| 6801 4439 \| 6540 200 \| 200 100 \| 100 734 \| 734 26.3, 25.8 \| 26.2, 26.4 10^4 3856 \| 8289 3829 \| 7890 200 \| 200 100 \| 100 734 \| 734 28.9, 27.8 \| 29.1, 29.3 10^3 164 \| 9484 157 \| 9061 157 \| 200 100 \| 100 734 \| 734 31.4, 31.1 \| 32.7, 32.8 10^2 94 \| 4939 85 \| 4725 85 \| 200 100 \| 100 734 \| 734 34.2, 34.8 \| 34.8, 35.3 10^1 133 \| 2652 131 \| 2520 131 \| 200 99.04 \| 99.86 729 \| 734 34.9, 34.8 \| 36.9, 36.7 aObtained from output of Porechop bObtained from output of LAclust cObtained from output of BWA-MEM. Input into BWA-MEM was limited to 200 reads based on LAclust options dConsensus sequence identity to the reference sequence of NDV LaSota sequenced with MiSeq (MH392212/chicken/USA/LaSota/1946) eEach dilution performed in duplicate and threshold cycle (Ct) values from each well are shown here fRun 1 gRun 2 Note: All 60,000 reads obtained during 32 min of sequencing run (R1) were utilized for the analysis. All 98,916 reads from R2 were utilized for the analysis ble 1 Comparison of MinION sequencing to RT-qPCR for detection of NDV LaSota (runs 1 and 2) ained during 32 min of sequencing run (R1) were utilized for the analysis. All 98,916 reads from R2 were utilized for the analysis Run 2 Note: All 60,000 reads obtained during 32 min of sequencing run (R1) were utilized for the analysis. All 98,916 reads from R2 were ut in a single 12-h MinION run (run = 4) generating a total of 2.076 million reads. The first 100,000 reads, which were obtained in 3 h and 10 min, were analyzed for identifica- tion of all 33 NDV isolates used in the study. All 33 NDV isolates were correctly identified to the sub-genotype level (Table 3), with 97.82–100% sequence identity. Clinical swab samples from chicken To assess the potential utility of this protocol on field samples from disease outbreaks, MinION libraries were generated directly from clinical swab samples. These swab samples were also propagated in eggs and the al- lantoic fluid was sequenced using a MiSeq-based work- flow (runs 5, 6, and 7) to compare to the MinION results. Out of 11 NDV-positive samples with the MiSeq method, Phylogenetic analysis Thirty-one of thirty-three samples were greater than 99% identical to the expected genotype in each of the sample, with 22 of 33 having 100% sequence identity. Samples with higher se- quence identity represent those isolates whose sequences (Sanger or MiSeq based) had already been deposited in GenBank, while those samples with lower sequence identity lacked replicate sequences from those particular isolates. For sample #37, MiSeq detected genotypes XIIIb and VIc, but the AmpSeq workflow only detected geno- type XIIIb (e.g., see pairwise comparison section for further demonstration of this protocol’s accuracy). were tested. All AAvV-1 genotypes that are currently circulating globally were amplified with tailed primers; samples 19 and 36 had weak bands of the desired mo- lecular weight (i.e., 832 bp) compared to other lanes. Two bands larger than 800 bp were visible on the elec- trophoresis of samples #19, #20, #21, #31 and #32 (see Additional file 2: Figure S1 legend for interpretation of this result). All non-AAvV-1 viruses failed to produce bands visible by gel electrophoresis (see Additional file 2: Figure S1). Quality metrics Th Q The Nanopore QC tool was used to obtain quality metrics plots of all sequencing runs. For MinION runs 3 (6-sam- ple pool) and 4 (33-sample pool), more than 70% of total reads had a quality score greater than ten (Q10 score = 90% accuracy) (Fig. 2a and b). The average overall mean read quality scores in both runs were comparable (run 3 = 10.7, run 4 = 11.0), and the mean quality scores of reads ≥10 (mean Q≥10) were similar (11.8) for both runs (Fig. 2 C and D). In addition, analysis of five consecutive batches of reads (each batch = 20,000 reads) obtained at different time intervals from run 4 indicated that the overall mean read quality for each 20,000 read batch remained above 10 (see Additional file 3: Table S4). Similarly, the mean Q≥10 over time remained consistent in the clinical sample runs (runs 5–7), which had long (12 h) sequencing runs (Fig. 3a, b and c, blue lines). While 832 bp was the expected amplicon size, geno- types III, IV, and IX (all previously untested genotypes with this primer set) yielded an unexpected electrophor- esis product of ~ 1000 bp (see above). The analysis of sequences obtained from these NDV isolates revealed that in addition to the 788 bp adapter-trimmed consen- sus sequence, an upstream region of NDV genome was amplified, resulting in an 1067 bp adapter-trimmed consensus sequence that contains the targeted NDV sequence. Sub-genotypic resolution of AAvV-1 viruses with MinION sequencing The overall read quality average (●) remained above 10 in both runs 10 samples were NDV positive by the MinION protocol (Sample #52 being the exception) (Table 4). In the six NDV-positive samples that contained one NDV genotype, as detected by the MiSeq method, the same NDV geno- type was also detected with the MinION protocol. The MiSeq method detected two genotypes in samples #45, #46, #47, and #49; whereas, the MinION protocol only de- tected dual genotypes in samples #45 and #46. In sample #48, only one NDV genotype was detected by MiSeq but 10 samples were NDV positive by the MinION protocol (Sample #52 being the exception) (Table 4). In the six NDV-positive samples that contained one NDV genotype, as detected by the MiSeq method, the same NDV geno- type was also detected with the MinION protocol. The MiSeq method detected two genotypes in samples #45, #46, #47, and #49; whereas, the MinION protocol only de- tected dual genotypes in samples #45 and #46. In sample #48, only one NDV genotype was detected by MiSeq but two NDV genotypes were detected by the MinION proto- col. All 4 samples negative by MiSeq were also negative by the MinION protocol. Sub-genotypic resolution of AAvV-1 viruses with MinION sequencing To determine the capability to effectively detect and differentiate viruses of different genotypes and sub-ge- notypes, PCR amplicons from 33 egg-grown isolates, which were representative of 23 different NDV genotypes and sub-genotypes, were barcoded, pooled, and sequenced Page 7 of 14 Butt et al. Virology Journal (2018) 15:179 Table 2 Accuracy of consensus sequence from serial dilutions (EID50/ml) of NDV LaSota during MinION sequencing run 1 Sequencing run time (min) Total Raw reads 10^6 10^5 10^4 10^3 10^2 10^1 NDV readsa % Identityb NDV reads % Identity NDV reads % Identity NDV reads % Identity NDV reads % Identity NDV reads % Identity 5 4000 283 100 199 100 182 100 10 99.18 2 NA 3 NA 7 8000 644 100 406 100 368 100 14 99.32 8 98.77 5 98.37 10 12,000 1029 100 661 100 571 100 23 99.32 16 99.32 8 99.18 12 16,000 1397 100 901 100 759 100 29 99.86 17 99.32 14 99.45 14 20,000 1772 100 1173 100 1014 100 36 99.73 20 99.45 17 99.59 16 24,000 2183 100 1451 100 1231 100 43 99.73 23 99.45 21 99.59 19 28,000 2643 100 1775 100 1498 100 56 99.86 29 99.73 25 99.45 aThe numbers represent total number of NDV reads obtained from LAclust. A maximum of 200 reads (optional cut-off value) were used to generate full length consensus sequence. Minimum 5 reads were used as a cut-off to build consensus sequence bConsensus sequence identity to the reference sequence of NDV LaSota sequenced with MiSeq (MH392212/chicken/USA/LaSota/1946) Consensus sequences were BLAST searched against NDV custom database Note: Only 28,000 out of total 60,000 reads were utilized for the analysis Butt et al. Virology Journal (2018) 15:179 Page 8 of 14 Fig. 2 Quality metrics of two MinION sequencing runs. Mean read-based quality score distribution of 6 sample pooled run (run 3) (a) and 33 sample pooled run (run 4) (b). Mean run-based quality score over time of six sample pooled run (c) and 33 sample pooled run (d). The overall read quality average (●) remained above 10 in both runs Fig. 2 Quality metrics of two MinION sequencing runs. Mean read-based quality score distribution of 6 sample pooled run (run 3) (a) and 33 sample pooled run (run 4) (b). Mean run-based quality score over time of six sample pooled run (c) and 33 sample pooled run (d). Pairwise comparison of replicated MinION sequences and MiSeq sequences Pairwise nucleotide distance analysis was used to com- pare the consensus sequences in six samples across two separate MinION runs. There was no variation in the Fig. 3 Mean Q ≥10 (blue lines) and Q < 10 (orange lines) of total reads over time during MinION sequencing runs using clinical swab samples. Additionally, the overall read quality average (●) for all three runs, remained above 10. a: MinION run 5, n = 9 samples, runtime = 6 h. b: MinION run 6, n = 4 samples, runtime = 7 h. c: MinION run 7, n = 2 samples, runtime = 12 h Fig. 3 Mean Q ≥10 (blue lines) and Q < 10 (orange lines) of total reads over time during MinION sequencing runs using clinical swab samples. Additionally, the overall read quality average (●) for all three runs, remained above 10. a: MinION run 5, n = 9 samples, runtime = 6 h. b: MinION run 6, n = 4 samples, runtime = 7 h. c: MinION run 7, n = 2 samples, runtime = 12 h Butt et al. Virology Journal (2018) 15:179 Page 9 of 14 consensus sequence between the MinION runs across those six samples. Pairwise nucleotide distance analysis was also used to compare the MinION consensus se- quence to the MiSeq consensus sequence in 24 isolates pairwise nucleotide comparison). Pairwise comparison of replicated MinION sequences and MiSeq sequences The MinION and MiSeq consensus sequences were 100% identical, except in four samples (#20, #25, #36, and #37), in which the percent identity was 99.18–99.86% (nota bene: the sam- Table 3 Identification and virulence prediction of NDV genotypes from 33 egg-grown samples (runs 3 and 4) Sample ID Input genotypea Output genotypeb BLAST search Alignment length Percent identity Fusion protein cleavage sitec, □ 1 II* II MH392212/chicken/USA/LaSota/1946 732 100 low virulent 2 II II KJ607167/LHLJ/2/goose/2006/China 734 100 low virulent 3 II II KJ607167/LHLJ/2/goose/2006/China 732 100 low virulent 4 II II EU289029/turkey/USA/VG/GA-clone_5/1987 734 99.86 low virulent 5 Ia* Ia MH392213/chicken/Australia/Queensland/V-4/10/1966 734 100 low virulent 15 Ia Ia MH392213/chicken/Australia/Queensland/V-4/10/1966 734 99.86 low virulent 16 VIId VIId KU295454/chicken/Ukraine/Lyubotyn/961/2003 735 99.46 virulent 17 II* II MH392228/poultry/Canada/Ontario/Berwick/853/1948 735 100 virulent 18 II II MH392228/poultry/Canada/Ontario/Berwick/853/1948 732 98.36 virulent 19 III* III MH392214/chicken/India/Mukteswar/519/1940 734 100 virulent 20 IV IV MH392215/chicken/Nigeria/Kano/1973/N52/899/1973 734 99.86 virulent 21 IV IV EU293914/Italy/Italien/1944 734 99.05 virulent 22 XIVb* XIVb KT948996/domestic_duck/Nigeria/NG-695/KG.LOM.11–16/2009 734 100 virulent 23 Va* Va MH392216/cormorant/USA/MN/92–40,140/250/1992 734 100 virulent 24 Vb* Vb MH392217/turkey/Belize/4338-4/607/2008 734 100 virulent 25 Vc* Vc MH392218/chicken/Mexico/NC/23/686/2011 733 99.73 virulent 26 VIc* VIc KY042125/chicken/Bulgaria/Dolno_Linevo/1992 734 100 virulent 27 VIm* VIm KX236101/pigeon/Pakistan/Lahore/25A/2015 734 100 virulent 28 VIIj* VIIj MH392219/chicken/Egypt/Sohag/18/1020/2014 734 100 virulent 29 VIIe VIIe KJ782375/goose/China/GD-QY/1997 734 97.82 virulent 30 VIIi* VIIi KX496962/ wild_pigeon/Pakistan/Lahore/20A/996//2015 734 100 virulent 31 IX* IX MH392220/poultry/China/04-23/C12/647/2004 734 100 virulent 32 IX IX MH392220/poultry/China/04–23/C12/647/2004 734 99.86 virulent 33 Xb* Xb MH392221/mallard/USA/MN/99–376/163/1999 734 100 low virulent 34 Xa* Xa GQ288378/ northern_pintail/USA/OH/87–486/1987 734 100 low virulent 35 XIIa* XIIa JN800306/poultry/Peru/1918-03/2008 734 100 virulent 36 XIIIb* XIIIb MH392222/chicken/Pakistan/SPVC/Karachi/27/558/2007 734 99.18 virulent 37d VIc/XIIIb* XIIIb MH392223/chicken/Pakistan/SPVC/Karachi/33/556-XIII/2007 734 99.46 virulent 38 XIVb* XIVb MH392225/chicken/Nigeria/KD/TW/03 T/N45/720/2009 734 100 virulent 39 XVI* XVI MH392226/chicken/Dominican_Republic/FO/499–31/505/2008 734 100 virulent 40 XVIIa* XVIIa KY171995/VRD124/06/N11/867/chicken/2006/Nigeria 734 100 virulent 41 XVII* XVII KU058680/903/domestic_duck/Nigeria/KUDU-113/1992 734 100 virulent 42 XVIIIb* XVIIIb MH392227/chicken/Nigeria/OOT/4/1/N69/914/2009 734 100 virulent aInput genotype was determined with MiSeq sequencing () or previous Sanger sequencing bDetermined by MinION sequencing cF protein cleavage sites of virulent NDV genotypes contains more than 3 basic amino acids [(112(R/K)-R-(Q/K/R)-(R/K)-R-F117)] and low virulent NDV genotypes has monobasic amino acids [(112(G/E)-(R/K)-Q-(G/E)-R-L117)] dIllumina Miseq detected two NDV genotypes Matching MiSeq result from same isolate □The fusion protein cleavage sites did not vary between AmpSeq and either previous Sanger or previous MiSeq Note: Isolates known to have low virulence are highlighted in bold pairwise nucleotide comparison). Pairwise comparison of replicated MinION sequences and MiSeq sequences Virology Journal (2018) 15:179 Table 4 Identification and virulence prediction of NDV genotypes in clinical samples collected during outbreaks in 2015 (run 5, 6, and 7) Sample ID Miseq genotypes MinION genotypes ID of the MinION hit Reads/ cluster Consensus length Percent identity Fusion protein cleavage site□ 44 VIIi VIIi chicken/Pakistan/Wadana_Kasur/PNI_PF_(14F)/2015 200 734 100 virulent 45 VIIi II VIIi II chicken/Pakistan/Wadana_Kasur/PNI_PF_(14F)/2015 chicken/USA/LaSota/1946 28 5 734 733 99.31 96.44 virulent low virulent 46 VIIi II VIIi II chicken/Pakistan/Wadana_Kasur/PNI_PF_(14F)/2015 chicken/USA/LaSota/1946 10 17 733 733 99.13 98.51 virulent low virulent 47 VIIi II NDd II NAe chicken/USA/LaSota/1946 NA 139 NA 732 NA 99.32 NA low virulent 48 ND VIIi II VIIi chicken/USA/LaSota/1946 chicken/Pakistan/Wadana_Kasur/PNI_PF_(14F)v/2015 200 21 732 733 99.59 99.13 low virulent virulent 49 VIIi II ND II NA chicken/USA/LaSota/1946 NA 200 NA 732 NA 99.32 NA low virulent 50 VIIi VIIi chicken/Pakistan/Wadana_Kasur/PNI_PF_(14F)/2015 113 734 100 virulent 51 VIIi VIIi chicken/Pakistan/Mirpur_Khas/3EOS/2015 200 734 100 virulent 52a VIIi ND NA NA NA NA NA 53 VIIi VIIi exotic Parakeets/Pakistan/Charah/Pk29/29A/2015 5 726 98.5 virulent 54 NO NDV ND NA NA NA NA NA 55 NO NDV ND NA NA NA NA NA 56 NO NDV ND NA NA NA NA NA 57 NO NDV ND NA NA NA NA NA 58 VIIi VIIi chicken/Pakistan/Gharoo/Three_star_PF_(7G)/2015 8 729 99.32 virulent TNb NA ND NA NA NA NA NA ENc NA ND NA NA NA NA NA aAfter bead purification, the barcoded amplicon concentration of this sample was lowest in this pool bTemplate control negative cNegative extraction control dNot detected eNot applicable □The fusion protein cleavage sites did not vary between AmpSeq and previous MiSeq Note: Isolates known to have low virulence are highlighted in bold Table 4 Identification and virulence prediction of NDV genotypes in clinical samples collected during outbreaks in 2015 (run 5, 6, d ) green font) grouped together with the viruses that showed highest nucleotide sequence identity to them, including those in which MiSeq sequences were avail- able (red font). The six isolates that were sequenced twice (blue font) clustered together. Taken together, the results demonstrated that all sequences clustered to the expected genotype/sub-genotype branch of the phylogenetic tree. the percent identity may be low due to the exact isolate not having a representative sequence in GenBank, e.g., sample 21 with 99.05% similarity). Pairwise comparison of replicated MinION sequences and MiSeq sequences In addition, there were no differences at the fusion gene cleavage site be- tween AmpSeq and either previous Sanger or previous MiSeq results (Tables 3 and 4, last column). Collectively, these results demonstrate the repeatable high accuracy of the MinION-AmpSeq method. Pairwise comparison of replicated MinION sequences and MiSeq sequences The MinION and MiSeq consensus sequences were 100% identical, except in four samples (#20, #25, #36, and #37), in which the percent identity was 99.18–99.86% (nota bene: the sam- ples in Table 3 without asterisks did not have a second sequence directly from that stock for comparison; thus, consensus sequence between the MinION runs across those six samples. Pairwise nucleotide distance analysis was also used to compare the MinION consensus se- quence to the MiSeq consensus sequence in 24 isolates (one isolate representing each genotype and subgeno- type; 24 samples with asterisks in Table 3 were used for Page 10 of 14 Butt et al. Discussion Additionally, while the preliminary analytical sensitiv- ity of this protocol was determined using only one NDV genotype, the sensitivity of the MinION AmpSeq was comparable to the matrix RT-qPCR test, which does not allow inference of virulence. Further testing of the NDV AmpSeq sensitivity to current virulence-predicting RT-qPCR tests are warranted [14, 15]; however, even these tests lack the genotyping capability of AmpSeq. The ability of this AmpSeq method to detect different genotypes of NDV was further aided by barcoding PCR, which adds an- other round of PCR to the assay and the ability to adjust the concentration of samples during the library preparation phase. This latter step allows for more volume of low con- centration (i.e., weak positives) samples to be added to the library pool. While the additional steps for library synthesis provide these advantages, they also add time to the assay (see below for further discussion of time efficiency). How- ever, the benefit of implementing detection, genotype pre- diction, and virulence prediction into a single test adds value to this assay. This study describes the development of a single proto- col for rapid and accurate detection, virulence deter- mination, and preliminary genotype identification (with sub-genotype resolution) of NDV utilizing the low-cost MinION sequencer. Additionally, an assembly-based se- quence analysis workflow for MinION amplicon sequen- cing data was developed. This MinION AmpSeq workflow detected all currently circulating genotypes when using egg-grown viruses. Furthermore, clinical swab samples were used to demonstrate proof of concept that such sam- ples contained sufficient NDV nucleic acid for detection, and interestingly AmpSeq detected 2 NDV genotypes (vaccine and virulent strains) in several clinical swab sam- ples. These capabilities suggest this protocol may be useful for research and ancillary diagnostic procedures and indi- cates that further development and validation of NDV AmpSeq would be useful, especially in developing coun- tries where NDV is endemic and there is a need for afford- able epidemiological surveillance to track reservoirs and disease outbreaks. The sequence heterogeneity among AAvV-1 genomes, which hinders the ability to develop a single test that sensitively detects NDV while also predicting the geno- typic classification and virulence, is well known [4, 51, 52]. Currently, an RT-qPCR targeting the M gene [10] is most sensitive and is used for screening samples, but this assay only provides positive and negative results of the samples. Discussion RT-qPCRs that predict virulence based on the fusion gene are available [10, 54]; however, the lower sensitivity of these assays and the inability of at least one of these assays to detect viruses of all geno- types (e.g., genotypes Va and VI) [10, 12, 13] complicate diagnostic interpretation when the matrix and fusion tests have conflicting results. Thus, the only truly reli- able option to detect a broad range of viruses and to determine virulence from some strains is to design multiple tests that include genotype-specific primers and probes [7, 12, 53]. Recently, Miller et al. reported that the primer set used in this study detected Class I and all nine of the tested class II genotypes [38]; however, this primer set was not tested against other currently cir- culating genotypes. The current study includes six add- itional genotypes, collectively representing all currently circulating genotypes (excluding the Madagascar-limited genotype XI). Furthermore, the ability to use AmpSeq as the final measure of a PCR allows for larger amplicon sizes as compared to RT-qPCR. As such, there will be one less restriction on primer site design when trying to create a While the multifaceted nature of this MinION AmpSeq protocol is an advantage, time and cost efficiency must be maintained for it to be useful. MinION is inherently rapid due to the real-time nature of the sequencing. For ex- ample, this method identified the correct NDV genotype in all serial dilutions, with an accuracy of 98.37–100%, after only 7 min of sequencing. Because the MinION pro- vides real-time sequence data, it is possible to monitor the sequencing run to determine the optimal run length for each library. Additionally, samples can be multiplexed into a single sequencing run, which reduces time and cost [55]. Recently, multiplexing and MinION sequencing of the PCR products from a panel of 5 samples was reported [55]. Here a panel of 33 samples was multiplexed while maintaining successful NDV genotyping from data col- lected within 3 h and 10 min of sequencing and without affecting mean read quality and percentage of high-quality reads. Thus, this protocol provides the flexibility to rapidly and economically obtain accurate sequence data for a pre- liminary genotyping and virulence prediction. While Nanopore sequencing has numerous benefits, the high error rate poses unique challenges to data ana- lysis. Thus, it is important to extract accurate consen- sus sequences from raw sequencing data [56]. Time and cost estimation The time of sample processing and cost estimation of re- agents to multiplex and sequence samples (n = 6; n = 33) from RNA extraction to obtain final consensus sequences is presented in Additional file 3: Table S5. From RNA extraction to final consensus sequence calculations, the average time (including sequencing time) to process six samples was approximately 9–10 person-hours and for 33 samples approximately 26 person-hours. Assuming that flow cell can be used multiple times (twice when 33 sam- ples pooled and five times when six samples pooled to To confirm the ability of the MinION-acquired partial matrix and fusion gene sequences to be used for accurate analysis of evolutionary relatedness, phylogenetic analysis using consensus sequences (734 bp; trimmed of adapter and primer sequences) obtained from two independent MinION runs (run 3 and 4) was performed. Additionally, the 24 sequences from MiSeq were also included in the phylogenetic tree (Additional file 4: Figure S2, to further illustrate the agreement between these two sequencing methods. In the phylogenetic tree, the isolates (n = 33; Page 11 of 14 Butt et al. Virology Journal (2018) 15:179 Page 11 of 14 Page 11 of 14 pan-NDV primer set. Work is in progress to utilize the ability of MinION to sequence longer amplicon frag- ments, which will provide more complete phylogenetic in- formation. After optimizing pan-NDV primer design for AmpSeq, sensitivity of pan-NDV AmpSeq will need to be further evaluated. prepare one cDNA library) for sequencing, cost per sequencing run and cost per sample were estimated. The cost per sample decreased from $53 (six samples multi- plexed) to $31 (33 samples multiplexed). Conclusions the noisy reads currently produced by the MinION plat- form. The approach in this study takes advantage of the fact that single MinION reads often represent full-length amplicon sequences. By clustering full-length reads based on pairwise identity and subsequently performing consen- sus calling using standard multiple alignment software, this method quickly and reliably generates accurate (con- sistently greater than 99% sequence identity to paired MiSeq) de novo assemblies from amplicon datasets using as few as twenty reads per amplicon, and correct geno- typic prediction with as few as five reads per amplicon. Thus, this approach overcomes the inherently high error rate (~ 90% accuracy) of Nanopore sequencing [57] and sequence identification and differentiation at the sub-genotype level can be highly reliable. Taken together, this protocol reliably detected, genotyped, and predicted the virulence of NDV using laboratory stocks of all genetic variants currently circulating worldwide. Fur- thermore, preliminary testing of clinical-based samples sug- gests its feasibility using clinical swab samples. This assay can be used for research purposes and as an ancillary test in field investigations; however, further testing, including sensitivity validation on clinical samples and testing the ef- fect of multiple isolates on sensitivity are warranted. Fur- thermore, the advantages of MinION AmpSeq allow for further optimization not possible with other techniques. For example, PCR product length is less of a restriction with MinION AmpSeq as compared to RT-qPCR. Overall, MinION AmpSeq improves the depth of information ob- tained from PCRs and allows for more flexibility in assay design, which can be broadly applied to the detection and characterization of numerous infectious agents. One known source of error in Nanopore sequencing is that 5-mers of A and T in the individual reads are difficult to identify accurately with MinION sequencing [58]. Importantly, a 5-mer run of a single base is not present in the cleavage site of the Fusion gene, however, two positions on the consensus sequence where there are 5-mers of A and C are present. Because of 2 in- stances in which only 4 nucleotides were read (one two nucleotide gap at 153–57 bp position and second on 233 bp) on 5-mer site, sample #37 had less than 100% identity to the respective Miseq data. Conclusions It should be noted that this type of system error can be easily detected and manually corrected for a paramyxovirus (including NDV), which are viruses that do not tolerate single nucleotide de- letions or insertions (rule of six) [59]. Because of the consensus-based approach, despite the relatively high, read-based error rate, the cleavage site was accurately determined. Additional files Phylogenetic tree constructed by using the nucleotide sequence (734 bp) of NDV isolates sequenced with MinION and MiSeq, with sequences of related NDV genotypes from GenBank. The evolutionary histories were inferred by using the maximum-likelihood method based on General Time Reversible model with 500 bootstrap replicates as implemented in MEGA 6. The tree with the highest log likelihood (−9347.8021) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories [+G, parameter = 0.9254]). The percentages of trees in which the associated sequences clustered together are shown below the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analyses involved 129 nucleotide sequences with a total of 725 positions in the final datasets. The sequences obtained in the current study are denoted with solid circles in front of the taxa name and bold font. Blue circles indicate isolates from MinION sequen- cing run 1, green circles indicate isolates from MinION sequencing run 2 and red circles indicate MiSeq sequencing. (PPTX 81 kb) Additional file 4: Figure S2. Phylogenetic tree constructed by using the nucleotide sequence (734 bp) of NDV isolates sequenced with MinION and MiSeq, with sequences of related NDV genotypes from GenBank. The evolutionary histories were inferred by using the maximum-likelihood method based on General Time Reversible model with 500 bootstrap replicates as implemented in MEGA 6. The tree with the highest log likelihood (−9347.8021) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (4 categories [+G, parameter = 0.9254]). The percentages of trees in which the associated sequences clustered together are shown below the branches. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analyses involved 129 nucleotide sequences with a total of 725 positions in the final datasets. The sequences obtained in the current study are denoted with solid circles in front of the taxa name and bold font. Blue circles indicate isolates from MinION sequen- cing run 1, green circles indicate isolates from MinION sequencing run 2 and red circles indicate MiSeq sequencing. (PPTX 81 kb) Discussion As previously discussed, pathogen typing from sequencing data can be done with read count-based profiling or de novo assembly approaches [31]. However, there are a limited number of available tools suitable for handling Page 12 of 14 Butt et al. Virology Journal (2018) 15:179 Butt et al. Virology Journal (2018) 15:179 Additional files Additional file 1: Table S1. The representative genotypes of AAvV-1 and other AAvVs used in this study (egg-grown viruses). Table S2. Background information of clinical swab (oral and cloacal) samples collected from chicken during disease outbreaks in Pakistan in 2015. Table S3. Detail of MinION sequencing runs. (DOCX 35 kb) Additional file 2: Figure S1. Agarose gel electrophoresis of AAvVs. Samples 6–14 and 43 are AAvVs other than AAvV-1. A DNA ladder (100 bp) was loaded into lane L. A no-template control was loaded into lane N. Bright bands show the amplified target region of AAvA-1 genome (expected product size 832 bp). See Table S1 for key to lanes. An unex- pected product of approximately 1100 bp was identified in samples #19, #20, #21, #31, and #32. Analysis of these genomes identified a second potential primer binding site. Additionally, two consensus sequences were obtained from these samples. One was the expected amplicon, and the second was a 1067 bp sequence that corresponded to the predicted second primer binding site (data not shown) and included the targeted amplicon sequence. (DOCX 840 kb) Because this protocol relies on identity-based clustering prior to assembly, it maintains the ability to detect sam- ples with mixed NDV genotypes. For example, in this study four clinical samples had two different genotypes as detected by MiSeq analysis, two of which were correctly identified by the MinION AmpSeq workflow. In a fifth case, a mixed sample was detected by MinION AmpSeq, but not by MiSeq. A potential explanation for these differ- ences could be that the MiSeq sequencing was performed on egg-amplified samples, which may have altered the relative levels of the two genotypes, as compared to the direct clinical swab sample used for MinION sequencing. Additionally, the differences in molecular techniques (i.e., MinION: targeted; MiSeq: random) may have altered the relative abundance of the genotypes within the sequencing libraries. While further studies into the ability of this workflow to sensitively detect and differentiate NDV in samples with more than one genotype are ongoing, rapid NDV genotyping from clinical samples without culturing the virus in SPF eggs has the potential to facilitate disease diagnostics. Additional file 3: Table S4. Time-based quality metrics of MinION se- quencing run 4 (n = 33). Table S5. Estimation of cost of reagents and sample processing time for MinION sequencing. (DOCX 25 kb) Additional file 4: Figure S2. 1. Miller PJ, Koch G. In: Swayne DE, Glisson JR, Mcdougald LR, Nolan LK, Suarez DL, Nair VL, editors. Newcastle disease. Diseases of Poultry. 13th ed. Ames: John Wilkey and Sons, Inc; 2013. p. 89–107. Competing interests Th h d l h y y 15. FLU-LAB-NET. https://science.vla.gov.uk/flu-lab-net/docs/pub-protocol-avian- avulavirus-mole-pathotyp.pdf. Accessed 24 Sept 2018. avulavirus-mole-pathotyp.pdf. Accessed 24 Sept 2018. 16. Ambardar S, Gupta R, Trakroo D, Lal R, Vakhlu J. High throughput sequencing: an overview of sequencing chemistry. Indian J Microbiol. 2016; 56:394–404. Funding This project was supported by USDA National Institute of Food and Agriculture Grant no. 2018–67015-28306, USDA CRIS 6040–32000-072, DTRA grant 58.0210.5.006 and partially funded by BAA project [grant#685/FRCALL 12–6-2-0015], by Biosecurity Engagement Program, Bureau of International Security and Nonproliferation, at the U.S. Department of State and University of Georgia Research Foundation, Inc., Faculty Research Grants Program. 6. Aldous E, Mynn J, Banks J, Alexander D. A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathol. 2003;32:237–55. 7. Kim LM, King DJ, Guzman H, Tesh RB, da Rosa APT, Bueno R, et al. Biological and phylogenetic characterization of pigeon paramyxovirus serotype 1 circulating in wild north american pigeons and doves. J Clin Microbiol. 2008;46:3303–10. Abbreviations AAvV: Avian avulavirus; AICc: Akaike information criterion; AmpSeq: Amplicon sequencing; APMV-1: Avian paramyxovirus 1; BIC: Bayesian information criterion; EID50: 50% egg infectious dose; MAFFT: Multiple Alignment using Page 13 of 14 Page 13 of 14 Page 13 of 14 Butt et al. Virology Journal (2018) 15:179 Butt et al. Virology Journal (2018) 15:179 2. Amarasinghe GK, Ceballos NGA, Banyard AC, Basler CF, Bavari S, Bennett AJ, et al. Taxonomy of the order mononegavirales: update 2018. Arch Virol. 2018:1–12. Fast Fourier Transform; ND: Newcastle disease; NDV: Newcastle disease viruses; ONT: Oxford Nanopore Technologies; RT-qPCR: Reverse transcriptase- quantitative polymerase chain reaction; SEPRL: Southeast Poultry Research Laboratory; SPF: Specific pathogen free Fast Fourier Transform; ND: Newcastle disease; NDV: Newcastle disease viruses; ONT: Oxford Nanopore Technologies; RT-qPCR: Reverse transcriptase- quantitative polymerase chain reaction; SEPRL: Southeast Poultry Research Laboratory; SPF: Specific pathogen free 3. Nagai Y, Klenk H-D, Rott R. Proteolytic cleavage of the viral glycoproteins and its significance for the virulence of Newcastle disease virus. Virology. 1976;72:494–508. Availability of data and materials The sequences obtained in the current study were submitted to GenBank and are available under the accession numbers from MH392212 to MH392228. 8. Diel DG, da Silva LH, Liu H, Wang Z, Miller PJ, Afonso CL. 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Infect Genet Evol. 2016;39:22–34. We gratefully acknowledge Tim Olivier for technical assistance. Special thanks to the Fulbright U.S. Student Program for sponsoring Dr. Salman’s PhD research work. 5. Commission IOoEBS, Committee IOoEI. Manual of diagnostic tests and vaccines for terrestrial animals: Mammals, birds and bees: Office international des épizooties; 2008. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 17. Rhoads A, Au KF. Pacbio sequencing and its applications. Genomics Proteomics Bioinformatics. 2015;13:278–89. Consent for publication Consent for publication Not applicable. 14. Fuller CM, Brodd L, Irvine RM, Alexander DJ, Aldous EW. Development of an l gene real-time reverse-transcription pcr assay for the detection of avian paramyxovirus type 1 rna in clinical samples. Arch Virol. 2010;155:817–23. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. Authors’ contributions S. L. Butt extracted RNA from egg-grown isolates and clinical samples, created the MinION libraries, analyzed the MinION data, conducted the phylogenetic analyses, and wrote the manuscript. T. L. Taylor helped with the RT-qPCR and in creation and sequencing of the MinION libraries. J. D. Volkening developed the MinION data analysis workflow and assisted with manuscript preparation. K. M. Dimitrov contributed to the preparation and analysis of sequencing data, the phylogenetic analyses, and manuscript preparation. D. Williams-Coplin prepared NGS libraries. K. K. Lahmers assisted in data analysis and manuscript preparation, A. M. Rana provided clinical swab samples. D. L. Suarez assisted in data interpretation and manuscript preparation. C. L. Afonso and J. B. Stanton were involved in the design of the study, data analysis, data interpretation, and writing of the manuscript. All authors were involved with editing the manuscript. All authors read and approved the final manuscript. 9. Kim LM, King DJ, Suarez DL, Wong CW, Afonso CL. Characterization of class i Newcastle disease virus isolates from Hong Kong live bird markets and detection using real-time reverse transcription-pcr. J Clin Microbiol. 2007;45: 1310–4. 10. Wise MG, Suarez DL, Seal BS, Pedersen JC, Senne DA, King DJ, et al. Development of a real-time reverse-transcription pcr for detection of Newcastle disease virus rna in clinical samples. J Clinl Microbiol. 2004;42: 329–38. 11. Kim LM, Afonso CL, Suarez DL. Effect of probe-site mismatches on detection of virulent Newcastle disease viruses using a fusion-gene real-time reverse transcription polymerase chain reaction test. J Vet Diagn Investig. 2006;18: 519–28. 12. 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https://openalex.org/W4362618289	https://figshare.com/articles/journal_contribution/Supplementary_Table_2_from_Concurrent_Radiotherapy_and_Ipilimumab_Immunotherapy_for_Patients_with_Melanoma/22536880/1/files/40000180.pdf	English	null	Supplementary Table 1 from Concurrent Radiotherapy and Ipilimumab Immunotherapy for Patients with Melanoma	null	2,023	cc-by	222	ab a d ad at o t e apy o pat e ts t u esectab e a d/o etastat c e a o a Eligible patients (planned number for enrollment) Lead Center Treatment Details Clinical Trial Identifier age ng Unresectable metastatic melanoma, failed one prior systemic therapy (20) Stanford University Induction ipilimumab at 3 mg/kg every 3 weeks, for 4 treatments Radiation therapy starting 2 days after first ipilimumab dose. NCT01449279 ase I y - in table x) Unresectable locally advanced or metastatic melanoma with at least one melanoma metastasis accessible to radiation therapy (30) Institut Gustave Roussy Induction ipilimumab at 10 mg/kg every 3 weeks, for 4 treatments, and then every 12 weeks. Radiation therapy dose escalated from 9 Gy to 24 Gy in 3 fractions (4 dose levels) NCT01557114 With Metastatic melanoma with brain metastasis (24) Thomas Jefferson University Induction ipilimumab at different doses every 3 weeks, for 4 treatments. Radiation therapy is either whole brain (Arm A) and stereotactic radiosurgery (Arm B). NCT01703507 ation ody tic Metastatic melanoma, previously treated or untreated (6-26) University of Pennsylvan ia Ipilimumab and stereotactic body radiation therapy NCT01497808 umab and otactic ablative on therapy NCT01565837 ion ipilimumab at kg every 3 weeks, reatments, with or ut radiation py (30 Gy in 5 ns over 1 week, g 3 days before umab) NCT01689974
https://openalex.org/W4287709200	https://zenodo.org/records/3956750/files/7.Mahotina.I.pdf	Russian	null	КАЛЕНДАРНАЯ ОБРЯДНОСТЬ ЦЫГАН-ВЛАХОВ И СЭРВОВ ВО ВТОРОЙ ПОЛОВИНЕ ХХ – НАЧАЛЕ XXI в.2	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	7,980	Илона МАХОТИНА, Януш ПАНЧЕНКО1 Илона МАХОТИНА, Януш ПАНЧЕНКО1 Rezumat Ritualuri calendaristice a țiganilor-vlahi și servilor în a doua jum. a sec. XX – înc. sec. XXI Rezumat Ritualuri calendaristice a țiganilor-vlahi și servilor în a doua jum. a sec. XX – înc. sec. XXI Rezumat Ritualuri calendaristice a țiganilor-vlahi și servilor în a doua jum. a sec. XX – înc. sec. XXI Articolul este consacrat descrierii ritualurilor calen daristice a țiganilor-vlahi și servilor, conform unor sur se narative și vizuale. Țiganii-vlahi sunt printre cele mai puțin studiate grupuri de țigani. Până nu demult, nu s-au făcut studii despre structura lor socială și cultură. Arti colul conține o scurtă descriere etnografică a grupurilor etnice de romi-vlahi și servi, a activităților lor economice. Acest articol continuă publicațiile din 2014, 2018 și 2019 despre limba și cultura tradițională a țiganilor-vlahi și ser vilor, și reprezintă primul studiu detaliat al ritualurilor lor calendaristice. Autorii introduc materiale de teren inedite în circulația științifică. Articolul conține mostre de vorbi re, poezii rituale și calendaristice, dorințe tradiționale de noroc și toaste în limba ucraineană și romani. Articolul analizează mâncărurile tradiționale de sărbătoare: colivă, izvar, pască, colțunași; ceremonii menite să asigure bunăs tarea familiei; predicții despre soartă etc. Unele ritualuri și sărbători sunt descrise pentru prima dată în contextul culturii țigănești. În baza studiului au fost puse materiale le etnografice de teren colectate în 2013–2020 în regiunea Herson, Lviv, Odesa din Ucraina, regiunea Belgorod, Sta vropol și ținutul Krasnodar al Federației Ruse. Ключевые слова: цыгане, влахи, сэрвы, кален дарные праздники, календарная обрядность, обычаи. Summary Ritual calendar of Roma Vlaxurja and Servurja in the second half of the 20th century – in the early 21st centuryh The article is devoted to the description of the ritual calendar of Vlaxurja and Servurja Roma based on narra tive and visual sources. The Vlaxurja are among the least studied groups of Roma. Until recently, little research has been material culture of Vlaxurja and Servurja. The arti cle contains a brief ethnographic description of the Roma Vlaxurja and Servurja groups and their occupations. The article continues the publications of 2014, 2018, and 2019 on the language and traditional culture of the Vlaxurja and Servurja and it is the first detailed study of their cal endar rituals. The authors introduce unique field materials into scientific circulation. The article contains samples of speech, ritual poetry, traditional well-wishes and toasts in Ukrainian and Gypsy. The article analyzes the traditional ritual celebratory dishes: kutya, uzvar, Easter cake, dump lings, etc; rituals aimed at ensuring the well-being of the family; predicting the fate, etc. Some rituals and holidays are first described in the context of gypsy culture. The investigations based on the field ethnographic materials collected in 2013–2020 in Kherson Oblast, Lviv Oblast, Odessa Oblast of Ukraine, Belgorod Oblast, Stavropol Krai and Krasnodar Krai of Russia. Cuvinte-cheie: țigani, vlahi, servi, sărbătorile calen daristice, ritualurile calendaristice, obiceiurile. КАЛЕНДАРНАЯ ОБРЯДНОСТЬ ЦЫГАН-ВЛАХОВ И СЭРВОВ ВО ВТОРОЙ ПОЛОВИНЕ ХХ – НАЧАЛЕ XXI в.2 DOI: doi.org/10.5281/zenodo.3956750 вые описываются в контексте цыганской культуры. В основу исследования легли полевые этнографические материалы, собранные в 2013–2020 гг. в Херсонской, Львовской, Одесской обл. Украины, Белгородской обл., Ставропольском и Краснодарском крае РФ. REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE 2020, Volumul XXVII 54 E-ISSN: 2537-6152 Календарная обрядность цыган-влахов и сэрвов во второй половине XX – начале XXI в. Принадлежность к кочевым сэрвам считается поводом для гордости. Представители этих се мей чаще используют в быту цыганский язык и русско-украинский суржик и лучше сохранили традиционную культуру. Городские сэрвы так же общаются на суржике, но можно встретить и русскоязычные семьи. Кочевые и городские сэрвы редко проводят совместные мероприятия и вступают в брак. В зависимости от ситуации, понятие ғородськи́ сэ́рвы (или ғородськи́ цығаны) может быть употреблено в укор по отношению к представителям общины, отошедшей от тради ционного уклада жизни. Влахи относятся к наименее изученным группам цыган. Исследований, посвященных их социальной структуре и культуре, до недавнего времени не было. Не исследован вопрос о меж- групповых связях цыган. Данные мемуарных, литературных и других источников, относящие ся ко времени, когда традиция описания этниче ских и локальных групп еще не сформировалась, нуждаются в соотнесении с конкретными этни ческими общностями. Настоящая статья продолжает публикации 2014, 2018 и 2019 гг. [4, 5, 6, 7, 8], посвященные языку и традиционной культуре влахов и сэрвов. В основу исследования легли полевые этногра фические материалы, собранные в 2013–2020 гг. в Херсонской, Львовской, Одесской обл. Украи ны, Белгородской обл., Ставропольском и Крас нодарском крае РФ. Сэрвы хорошо интегрированы в окружение. Получают среднее и высшее образование, овла девают современными профессиями. В недавнем прошлом занятия сэрвов составляли барышни чество лошадьми, кузнечное и сапожное дело. у Цыгане-влахи5 (влáхуря) принадлежат к старожильческому слою цыганского населения Украины и юга России. Влахи входят в число эт ногрупп, наименее адаптированных к культуре окружения. Сохранили традиционные инсти туты. В настоящее время в районах наибольшей концентрации влахи проживают компактными поселениями [9, с. 22]. О прежнем полуоседлом образе жизни напоминает второе самоназва ние влахов – катунáря ‘шатёрники’. До выхо да Постановления Совета Министров РСФСР «О приобщении к труду цыган, занимающихся бродяжничеством» (1956 г.) влахи кочевали, до сих пор сохранив высокую мобильность уклада, связанную с необходимостью выезжать на зара ботки за пределы страны или региона прожива ния. Тесные культурные контакты влахов и сэр- вов, в форме как совместного кочевания, так и межгрупповых браков, препятствовали, на сколько мы можем судить, развитию значитель ной культурной дистанции между ними. Фак тически различия не настолько велики, чтобы культуру этих групп нельзя было рассматривать в рамках одного исследования. Полученные нами сведения относятся преимущественно к периоду перехода цыган к оседлости (середина XX в.). До недавнего времени традиционная куль тура влахов и сэрвов описана не была. В послед ние два десятилетия нерегулярно появлялись научные описания семейной обрядности сэр вов, проживающих в Украине и Средней Азии [11, 19]. Календарная обрядность цыган-влахов и сэрвов во второй половине XX – начале XXI в. Статья посвящена описанию календарной обряд ности цыган-влахов и сэрвов по нарративным и визу альным источникам. Влахи относятся к наименее изу ченным группам цыган. Исследований, посвященных их социальной структуре и культуре, до недавнего времени не было. В статье содержится краткое этно графическое описание этнических групп цыган-вла хов и сэрвов и их хозяйственных занятий. Настоящая статья продолжает публикации 2014, 2018 и 2019 гг., посвященные языку и традиционной культуре влахов и сэрвов, и представляет собой первое подробное ис следование их календарной обрядности. Авторы вво дят в научный оборот уникальные полевые материа лы. Статья содержит образцы речи, календарно-об рядовой поэзии, традиционные благопожелания и то сты на украинском и цыганском языке. В статье про анализированы традиционные праздничные блюда: кутья, взвар, пасха, вареники; обряды, направленные на обеспечение благополучия семьи; предсказания судьбы и др. Некоторые обряды и праздники впер Key words: Roma people, Vlaxurja, Servurja, ritual calendar, national holidays, rituals. Сэ́рвы (сэ́рвуря) – наиболее ранний и много численный слой цыганского населения Украины и юга РФ, в формировании диалекта и особенно стей культуры которого существенна роль укра инского окружения. Проживают как дисперсно, так и компактными поселениями. Сэрвы сохра нили родовое структурирование, традиционные этнические институты (в частности, этнический суд) и практики, связанные с представлениями об осквернении3. Родовая самоидентификация частично утрачена [9, с. 22]. Часть сэрвов жила оседло уже в середине XVIII в. В настоящее время сэрвы компактными REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE E-ISSN: 2537-6152 2020, Volumul XXVII 55 астраханские, донские и поволжские / волжские (пово́лжцуря)6. В уточнении нуждаются получен ные нами данные о существовании подгрупп ли нейских и бахмутских влахов. поселениями проживают практически во всех регионах Украины, а также в Калужской, Самар ской, Воронежской, Ростовской и других обла стях Российской Федерации. поселениями проживают практически во всех регионах Украины, а также в Калужской, Самар ской, Воронежской, Ростовской и других обла стях Российской Федерации. К традиционным хозяйственным занятиям влахов относятся кузнечество и лудильное дело, кустарное изготовление сельскохозяйственного инвентаря; плетение корзин; столярное дело; ба рышничество лошадьми и др. Женщины помо гали мужчинам в реализации товара, как сейчас помогают в сборе металлолома. Основными же вспомогательными занятиями, практикуемыми женщинами, традиционно были попрошайниче ство и гадание [7, c. 23]. Выделяются территориальные подразделе ния сэрвов, среди которых: воронежские (хан джя́ря), поволжские, полтавские (полтавци́), таврические (таврыча́ны), заднепрянские (за днипря́ны), кылмыши́. Помимо регионального, у сэрвов существует деление на городские (форо́стирэ сэ́рвуря / ғо родськи́ сэ́рвы) и кочевые4 (кочо́ви сэ́рвы) се мьи, что исторически связано с образом жизни рода, соответственно, оседлым или мобильным. Календарная обрядность цыган-влахов и сэрвов во второй половине XX – начале XXI в. гаджиканэ́ сэрбытóря, гаджéнски прáзныкы ‘не цыганские праздники’) – День защитника Отече ства, Восьмое марта, Новый год (влаш. кадá га жиканó нэ́во бэрш ‘это нецыганский Новый год’) и др. – цыгане не отмечают. Как правило, перед передачей икон семей ный сын (внук или младший брат) при свидете лях (среди которых его жена) должен от порога дойти до родителей на коленях. Обращаясь к старшим родственникам, хранителям фамиль ных икон (как правило, матери или бабушке), со словами: Мангáв тири́ блағослове́ния пэ бах та́тэ, пэ лоша́тэ тэ справисара́с о деса́ дэвли канэ́… [I] ‘Прошу твоего благословения на сча стье, на радость справлять нам дни божьи…’, мужчина трижды целует икону. Крупные праздники (Пасха, Рождество и т. д.) отмечаются обычно в течение трех дней. При ем гостей и визиты, наносимые в эти дни, свя заны с общественным положением и степенью родства визитеров и принимающей стороны. Обычно близких родственников и знакомых, обладающих в общине высоким авторитетом, приглашают или посещают в первый день празд ника. иконой и благословением он полу мечать праздники и приглашать го из традиций семьи, благословение ько на некоторые продукты, напри индюка, или на всё, кроме фруктов: вения́ дэ́л ай тэ авэ́л май-пхурэ́ тэ стон эрбыто́ря, ослове́ния, справи́н сэ а акхарэ́са шушо́ ска ке́ иси́ дэ́- ндо ка́жно по хамо́с. кай котэ́ ануша́ по по товэ́р и э и када́ за пхурэ́ндар . ‘Это благословение дает ся для того, чтобы было разрешение от старей ших людей ставить сто лы на праздники, если нет благословения, хозя ева не могут справлять праздники, потому что не позовешь же людей за пустой стол <...>. Еще есть некоторые законы у каждой семьи, не только на еду. Вот есть семья, там разговляются люди на топоре, становятся и так разговляются, и этот закон переходит от стари ков к молодым8. Вместе с иконой и благословением он полу чает право отмечать праздники и приглашать го стей. Исходя из традиций семьи, благословение могут дать только на некоторые продукты, напри мер, на хлеб и индюка, или на всё, кроме фруктов: Када́ блағословения́ дэ́л пэ ваш кода́, кай тэ авэ́л и розреше́ния май-пхурэ́ манушэ́ндар тэ стон скаминада́ пэ сэрбыто́ря, сар нина́й блағослове́ния, хулая́ наши́ тэ справи́н сэ рбыто́ря, бо на акхарэ́са жэ манушэ́н по шушо́ ска ми́нд <...>. Инке́ иси́ дэ́- савэ зако́нуря а́ндо ка́жно ери́, най кацы́ по хамо́с. А́кэ иси́ ери́, кай котэ́ розғови́йпэ мануша́ по товэ́р, тердё́н по товэ́р и ка́дя розғови́йпэ и када́ за ко́но пэрэжа́л пхурэ́ндар кай тэрнэ́ [ЯП]. Календарная обрядность цыган-влахов и сэрвов во второй половине XX – начале XXI в. Некоторые сведения содержатся в до революционных этнографических источниках и современной мемуаристике. Так, предвзятый взгляд на свадебный обряд цыган (предположи тельно, сэрвов) и цыганскую обрядность как та ковую отражен в этнографическом очерке Воро С историческим ареалом кочевания связа ны названия территориальных подразделений влахов, имеющих некоторые отличия в говоре и обычаях. Среди них выделяются кубанские (куба́нцуря), ставропольские (ставропóльцуря), REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE 2020, Volumul XXVII E-ISSN: 2537-6152 56 Тэ лэл блағослове́ния када́ тэ справи́й пра́зныкуря: о Крэчю́но, Патради́. Пэ́р во лэл и блағослове́ния о дад, пи́сля дад ашэ́лпэ лэ шавэ́сти акадэя́ вря́мя, май-цыкн́о шаво́, во залэ́л лэ дадэ́ско тхан. Лэ цыкнэ́ шавэ́сти на трэ́буй ни́со тэ мангэ́л, бо во бэшэ́л по пэсти дадэ́ско тхан. Ай барэдэ́р пхрал, о шаво́, пхэна́са, отжеля́ отде́ль но тэ жувэ́л, сар лэ́стэ иси́н жела́ния во мо́жэть тэ мангэ́л блағослове́ния кав пэ́ско дад, а́ли кав май-цыкно́ пхрал. Сар во дикхэ́л па пэ́сти са́ма, со во а́ндо зор кай тэ спра ви́й, тэ андярэ́л када́й вря́мя, ай сар на хутилэ́л возможно́сть, тоди́ на мангэ́н [III]. ‘Брать благословение – это праздновать празд ники: Рождество, Пасху. Первым берет благосло вение отец, после отца остается сыну это время7, младший сын занимает отцовское место. Млад шему сыну не нужно ни чего просить, потому что он живет на месте свое го отца. А старший брат, сын, скажем, отошел от дельно жить, если у него есть желание, он может просить благословения у своего отца или у млад шего брата. Если видит по своему состоянию, что он в силе праздновать, нести это время, а если не хватает возможности, тогда не просят’. нежской губернии второй половины XIX в. [10, с. 181]. В сборник цыганской поэтессы Р. Б. На барончук включен небольшой мемуарный очерк о Рождестве и текст колядки, исполнявшейся перед застольем в честь хозяина и хозяйки дома [17, с. 188-189]. Празднованию Рождества и Пас хи у украинских цыган посвящены материалы публицистов [3, 18]. Наиболее подробное на се годняшний день научное описание календарной обрядности влахов и сэрвов было обнародовано авторами этой статьи в коллективной моногра фии [4, с. 355-359]. Исторически влахи и сэрвы исповедуют православие. С 1990-х гг. в ряде регионов Укра ины и РФ отмечается переход цыганских семей в протестантизм [8, 9, с. 21], что может сказы ваться на сохранении обрядности. Календарная обрядность влахов и сэрвов связана с крупными религиозными, в данном случае православными, праздниками и в целом соотносится с традиция ми украинского и южнорусского окружения. Го сударственные праздники Украины и РФ (сэрв. Календарная обрядность цыган-влахов и сэрвов во второй половине XX – начале XXI в. ‘Это благословение дает ся для того, чтобы было разрешение от старей ших людей ставить сто лы на праздники, если нет благословения, хозя ева не могут справлять праздники, потому что не позовешь же людей за пустой стол <...>. Еще есть некоторые законы у каждой семьи, не только на еду. Вот есть семья, там разговляются люди на топоре, становятся и так разговляются, и этот закон переходит от стари ков к молодым8. Вместе с иконой и б чает право отмечать пра стей. Исходя из традици могут дать только на неко мер, на хлеб и индюка, ил Када́ блағословения́ дэ́л пэ ваш кода́, кай тэ авэ́л и розреше́ния май-пхурэ́ манушэ́ндар тэ стон скаминада́ пэ сэрбыто́ря, сар нина́й блағослове́ния, хулая́ наши́ тэ справи́н сэ рбыто́ря, бо на акхарэ́са жэ манушэ́н по шушо́ ска ми́нд <...>. Инке́ иси́ дэ́- савэ зако́нуря а́ндо ка́жно ери́, най кацы́ по хамо́с. А́кэ иси́ ери́, кай котэ́ розғови́йпэ мануша́ по товэ́р, тердё́н по товэ́р и ка́дя розғови́йпэ и када́ за ко́но пэрэжа́л пхурэ́ндар кай тэрнэ́ [ЯП]. Зимний цикл Утром 7 января некоторые цыгане, в основ ном женщины, отправляются в церковь. Вечером праздник продолжается новыми застольями. У сэрвов первый праздник зимнего цикла приходится на 19 декабря. День святителя Ни колая Чудотворца (сэрв. Дэнь Святоғо Николая, Мыколáйчик, Николáйчик) отмечают в семьях, где есть маленькие дети, которым под подушку кладут подарки. Родители маленьких сыновей с именем Николай накрывают небольшой стол и приглашают гостей. Те также приносят подарки для детей. р д р д Влахи отмечают Рождество большим общим застольем. Празднование также сопровождается обходом домов, в день обходят два или три дома. Один из обрядов этого вечера – разламывание на части и дележ праздничного индюка. Гости за ходят в дом со словами: Здра́вствуйте, хозя́ин с хозя́йкой, тэ авэ́н зоралэ́, тэ авэ́н бахталэ́ (будь те здоровы, будьте счастливы)! Разреши́ нам, пан хозя́ин, ва́шу пти́цу уби́ть! [VI] Гость раз ламывает тушку так, чтобы она приобрела фор му «кораблика»: разводит бедрышки, переднюю часть вырезает и поднимает, как «парус». При этом исполняют обрядовую песню «Поднялся корабль на раздутых парусах…». Бедро («писто лет») отдают хозяину (или другому уважаемому мужчине), второе – старшему сыну, крылышки – незамужним девушкам, переднюю часть режут на всех, гостям также отрезают по куску мяса. Похожий ритуал в данном регионе отмечен нами у нецыганского окружения [II]. Кроме индюка, на праздничном столе обязательно присутству ет пирог с творогом (пыри́жко тиралэ́са) или курагой (влаш. пыри́жко сушка́са / сэрв. пыро́ж ня9), иногда оба. Резавшая пирог женщина по лучает гузку индюка. Вместе пирог и индюк, по представлениям влахов, символизируют телегу и коня. Влахи начиняют индюка яблоками и рисом, подслащенным сахаром или изюмом. Современ Канун Рождества (Крэчю́но, в укр.-цыг. речи – Рожэствó) отмечается 6 января. В ком пактных поселениях цыган в Рождественский сочельник – Свят Вэ́чир, Вэчéря, Бағáто Вэ́че ро – дети не старше двенадцати лет (сэрв. вэчир ныкы́, влаш. свэнцомáря) носят вечерю (носят вэчерю). Родители собирают вечерникам кор зину с продуктами, среди которых обязательно присутствуют ритуальные постные блюда: рисо вая или пшеничная (Львов) кутья (кутя́) и взвар (узвáр). С наступлением сумерек с этой корзи ной дети начинают обходить дома родственни ков (малышей приводят родители), прежде все го крестных, со словами: Мáма и дáдо пэрэдáлы вам вэчéрю, тэ авэ́н тумэ́ бахталэ́, састэ́ и т. д. [ЯП] ‘Мама и папа передали вам вечерю, чтоб вы были счастливы, здоровы и т. д.’ Хозяева про буют и хвалят ритуальные блюда. Некоторые продукты из корзины оставляют себе, отдавая взамен свои. Детям дарят деньги или заранее приготовленные подарки. Праздничная еда. Вода для гостей У влахов праздничная еда называется вулай имо́с ‘неосвященная еда’ и свэнцомо́с ‘освящен ная еда’ [III]. Также в период любого праздника у ставропольских влахов на стол принято ставить сосуд с водой и стакан, из которого приходящие гости пьют «на удачу»: Кадя́ терэ́н, сар стон и меся́ля важ рромэ́ско зор, а́нда хамо́с баро́ де́ло на стон, кати́: о бо́ршо, ванза́ря, кру́мпля, ягали́, ай помашка́р ва́дра пайи́, <…> ай па́ша ва́дра тах та́ри. Стон ваш о зор меся́ля, кай ся дра́го тэ авэ́л <…> кай саворрэ́ндэ бах, зор тэ авэ́л. Хан, пэн, мангэ́нпэ Дэвлэ́с [VII]. ‘Так делают, как ста вят стол для здоровья цыгана, из еды большое дело не ставят, только: борщ, вареники, картош ку, водку, а посередине ведро воды, <…> а воз ле ведра стакан. <…> Ставят для здоровья стол, чтоб всё хорошо было <…> чтоб у всех счастье, здоровье было. Едят, пьют, просят Бога’. За столом ведутся беседы на религиозные темы. Более консервативные цыгане не расхо дятся до самого утра, у прочих застолье продол жается до двух-трех часов ночи. В этот день те, кто соблюдает пост, до наступления темноты не едят ничего скоромного, это называется ғоло́дна кутя́: Пи́сля шэсти́ вжэ наши́ тэ хас, хас кода́кэ, со по́стно, со пэ меся́ля иси́н. Пэ када́ вся хамо́с даши́ тэ пхэнэ́с вулайимо́с [III]. ‘После шести уже нельзя есть, ешь только что постное, что на столе есть. На эту всю еду можно сказать празд ничная неосвященная еда’. 2020, Volumul XXVII Данных о существовании подобного обряда у окружения или у цыган других групп нами не найдено. по родственникам считается уже неприличным. Позднее – около семи вечера – в доме накрывают стол. Гости приносят свои корзины со снедью, хозяин или хозяйка дома пробуют принесенный гостями узвар. Первую ложку кутьи хозяин под брасывает под потолок. Как и у окружения [15, с. 188], хорошей приметой считается, если часть ее приклеилась к потолку: это принесет счастье дому. Сидящие за столом гости-мужчины также пытаются поймать крупинки, что, по поверью, приносит удачу. Благословение Молодая влахицкая семья, начинающая жить отдельно от родителей мужа (тэ жал по отди́ло ‘идти на отдел’), получает возможность отмечать крупные религиозные праздники (Рождество, Пасху, Троицу, Спас) своим домом и приглашать гостей на торжество только после особого риту ала испрашивания благословения (тэ мангэ́л / тэ лэл блағослове́ния ‘просить / взять благосло вение’) и передачи им фамильных икон (тэ лэл масхари́ ‘взять икону’). Обряд, который приуро чивают к крупному празднику, Рождеству или Пасхе, проводится в семьях, где есть взрослые сыновья или внуки. Существование этого обы чая мы отмечаем у кубанских, ставропольских, астраханских и волжских влахов. Ритуал озна чает переход женатого человека в новый статус – главы семьи: REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE E-ISSN: 2537-6152 2020, Volumul XXVII 2020, Volumul XXVII 57 Обычай нашел отражение в рождественской колядке: Обычай нашел отражение в рождественской колядке: Разреши́те, пан хозя́ин, Вам коля́дку сказа́ть. Свят, свят да вэчо́р. Вам коля́дку сказа́ть, да Ваш дом весели́ть. Сэрвам известна традиция скармливания домашнему скоту ритуальной выпечки [13, c. 39, 42] (рожэ́ствэнык ‘рождественник, выпечка для коней’) в форме косички: Вам коля́дку сказа́ть, да Ваш дом весели́ть. Свят, свят да вэчо́р. Что твой двор, что мой двор на сто А на Рожэство́ ще пэ чу́ть, оцэ́ ки́сто сла́д кэ розка́туемо, и тоди́ бэрэ́м отако́ ро́бым, по луча́ется цэ спициа́льно для ко́нэй, про́сто кусо́к ки́ста, без начи́нкы, роз ка́чуэмо, цэ для ко́нэй, и то́жэ ложы́ться на по́куття. Озва́р до́лжэн обяза́тельно мужчы́на ста́выть, ша́пкы бэрэ́ й ста́вэ, и ло́жать и оци́ рожэ́ствэныкы для ко́нэй. Тоди́ як пра́зныкы одийшлы́, бэру́ть отэ́ си́но, оти́ рожэ́ствэны кэ и отдаю́ть худо́би, цэ на доста́ток, и коня́чка ску́шае. Ще ғолубци́ ро́ блять, ву́шка [IX]. и́ о к , а н а э́ и я ы э́ э а ‘А на Рождество еще пе кут, это тесто сладкое раскатываем, и потом берем вот так делаем, получается это специаль но для лошадей, просто кусок теста, без начин ки, раскатываем, это для лошадей, и тоже кладет ся на святой угол. Взвар должен обязательно муж чина ставить, шапки бе рет11 и ставит, и кладут эти рождественники для лошадей. Потом, когда праздники прошли, берут то сено, те рождественни ки и отдают скоту, это на достаток, и лошадка ску шает. Еще голубцы дела ют, ушки <вареники>’. Свят, свят да вэчор, А на э́тому двору́ стои́т столб золото́й, А на э́тому двору́ стои́т столб золото́й, Свят, свят да вэчо́р. А на э́тому двору́ стои́т пти́ца-орёл, Свят, свят да вэчо́р. Пти́ца царская ғосуда́рева, Свят, свят да вэчо́р. А по э́тому двору́ пан хозя́ин проходи́л, Свят, свят да вэчо́р. Лук и стре́лы вынима́л, хоте́л пти́цу уби́ть, ́ Лук и стре́лы вынима́л, хоте́л пти́цу уби́ть, Свят, свят да вэчо́р. Свят, свят да вэчо́р. «Ой, нэ бый ты мэнэ́, нэ стриля́й ты мэнэ́, Свят, свят да вэчо́р. Свят, свят да вэчо́р. Бу́дэш сы́на жэны́ть, до́чку за́миж отдава́ть, Свят свят да вэчо́р». А за эту коляду́ – ко́ня в ха́ту завэду́, Свят, свят да вэчо́р. По представлениям сэрвов, в ночь перед Рождеством нельзя подходить к конюшне. Счи тается, что после полуночи лошади начинают беседовать друг с другом, и подслушавший их разговор навлекает на себя беду: Дед розка́зу вав, шо оды́н цы́ған так пидслухо́вував ко́нэй и тоди́ заними́в [VIII]. Зимний цикл Вечерю носят и взрос лые, например, сын отцу, но подростку ходить REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE E-ISSN: 2537-6152 2020, Volumul XXVII 58 чер заводить в дом коня, что, по поверью, сулит хозяевам удачу и достаток: ные сэрвы могут запекать (в целлофановом па кетике) в ритуального индюка денежную купюру крупного достоинства, например, в пять тысяч рублей. уд у д На Рожэство́ заво́дять конэ́й, <...>. Пыта́ют ро зрише́ния: «Пан-хазя́ин, мо́жна завэсты́ у ха́ту коня́?». Спива́ють ко́ляд кы тоди́, цэ на добро́, на доста́ток – завэсты́ коня́ у ха́ту. Ось була́ лошы́ця, Ға́лка, завэлы́ до сто́лу йийи́, и вона́ оцэ́ со сто́лу йи́сть хлиб, шо там е, споко́йно сэбэ́ вэла́ [X]. у у На Рожэство́ заво́дять конэ́й, <...>. Пыта́ют ро зрише́ния: «Пан-хазя́ин, мо́жна завэсты́ у ха́ту коня́?». Спива́ють ко́ляд кы тоди́, цэ на добро́, на доста́ток – завэсты́ коня́ у ха́ту. Ось була́ лошы́ця, Ға́лка, завэлы́ до сто́лу йийи́, и вона́ оцэ́ со сто́лу йи́сть хлиб, шо там е, споко́йно сэбэ́ вэла́ [X]. ‘На Рождество заводят коней <в дом> <...>. Про сят разрешения: «Пан хозяин, можно завести в хату коня?» Поют коляд ки тогда, это на добро, на достаток – завести коня в хату. Вот была лошадка, Галка, завели к столу ее, и она вот так со стола ест хлеб, что там было, спо койно себя вела’. ть о н, ту д на ня́ я, лу лу е, ‘На Рождество заводят коней <в дом> <...>. Про сят разрешения: «Пан хозяин, можно завести в хату коня?» Поют коляд ки тогда, это на добро, на достаток – завести коня в хату. Вот была лошадка, Галка, завели к столу ее, и она вот так со стола ест хлеб, что там было, спо койно себя вела’. ру Взвар готовят в большой кастрюле, из кото рой его набирают для стола кувшином, наполняя его по мере необходимости. Пол под кастрюлей обязательно застилается сеном: Озва́р нико́лы нильзя́ ста́выть самы́й, цэ называ́еться – вин одовы́й, а кутя́ цэ як вона́, вжэ должна́ буть ко́ло ньо́го, и обяза́тельно си́но ло́жуть [IX]. ‘Взвар ни когда нельзя ставить один, это называется – он вдовый, а кутья это как она10, уже должна быть около него, и обязательно сено кладут’. Поста вив посуду с ритуальным блюдом на сено, глава семьи произносит: Узва́ро по база́ро, ай кутя́ пэ по́куття [ЯП]. ‘Узвар на базар, а кутья на святой угол’. 2020, Volumul XXVII 2020, Volumul XXVII В ночь с 13 на 14 января сэрвы отмечают Васильев вечер, называемый Романо Нэво бэрш ‘Цыганский Новый год’. В канун Цыганского Нового года – Малáнку, или Мэла́нку, – в ком пактных поселениях проходят ряжения и коля дование. Колядовать – мэланкува́ть – отправ ляются неженатые парни (ғольтя́я), девушки и дети. Ряженые – мэла́нкы – обходят дома все вместе или группами, в зависимости от общего числа и разницы в возрасте. Среди ряженых обя зательно присутствуют мальчик, изображающий Мэла́нку, и девочка, одетая в Ва́сылька. Для это го мальчику повязывают фартук, румянят щеки, девочке надевают картуз и мужскую куртку, по дрисовывают усы, дают в руки пастуший посох. вание может продолжаться до 7–9 часов утра. У некоторых семей сэрвов после посевания приня то посещать кладбище. Этим посещением, про ходящим скромно и тихо, заканчивается Цыган ский Новый год. На Мэланку и Крещение девушки гадают о судьбе и замужестве. Цыганские способы гада ния также заимствованы у восточнославянского окружения и практикуются, например, русскими цыганами [1, с. 93]. Девушки-мэланки во время колядования обращают внимание на первое сло во, услышанное от хозяина дома. По нему судят о своей судьбе. Вечером или ночью окликают прохожих. Жениха будут звать так же, как встре ченного незнакомца. По направлению следа бро шенного в снег ботинка примечают, в какую сто рону хозяйка обуви выйдет замуж. Обнимают штакетник, если число досок четное – выйдешь замуж в этом году. Под кровать ставят блюдце, на котором возводят мостик (из спичек), при говаривают: «Суженый, ряженый приди ко мне наряженный». Приснившийся человек станет мужем. Распространен способ гадания по тени от сгоревшей бумаги. Влахи также устраивали ряжения еще в пе риод полуоседлости. Девушки переодевались в мужскую одежду, которую брали у нецыган, так как цыганский мужчина не стал бы надевать ее после женщин. В цыганских домах прихода ряженых ожи дают за накрытыми столами. Ряженые поют ко лядки, принимают от хозяев денежные подноше ния (значительно меньшие, чем те, которые по лучают вечерники). В сэрвицкой среде известна колядка «Ой, вчора да з вэчора, пасла Мэланка два качора» (ср. тексты, записанные от окруже ния [16, с. 272]). У украинского окружения сэрвы также заимствовали следующий текст: у В цыганских домах не прижилась тради ция ставить елку. Это делают лишь в некоторых семьях с маленькими детьми. Весенний цикл Обычай нашел отражение в рождественской колядке: ‘Дед рассказывал, что один цыган так подслушивал коней и потом онемел’. Согласно данным других информантов, поверье распространяется на весь скот. По некоторым наблюдениям, ныне обычай трансформируется. Коня в собственный дом за водит хозяин или хозяйский сын. Многие семьи сохранили лишь память об обряде. Также до не давнего времени у сэрвов существовал шуточ ный обычай уводить на Рождество или Маланку у знакомых или родственников коня, которого потом возвращали за символический выкуп. На второй день праздника принято подавать ква́ско ‘квасок’ (крэчюнóско бóршо ‘рождествен ский борщ’). Лошадь могла использоваться в обряде рож дественского колядования. У сэрвов в ряде реги онов сохранился обычай в рождественский ве REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE E-ISSN: 2537-6152 59 Весенний цикл Весенний цикл начинается Масленицей, на которую принято выпекать блины, подаваемые с медом. На Вербное воскресенье (Вэ́рбна нэди́ля) ломают и освящают в церкви ветки вербы (лóзы); слегка ударяя ими детей, по-украински или по-цыгански произносят пожелание: влаш. Кадá най мэ марáв, ай вэрбá марэ́л! Тэ авэ́с ту бахталó, састó! Тэ фэри́й тут о Дэл! ‘Это не я бью, а верба бьет! Будь ты здоров, счастлив! Храни тебя Гос- подь!’ Известно пожелание: Сар марáв тут ла вэрбáса, тэ марэ́л о Дэл тут бахтáса! [VI] ‘Как бью тебя вербой, пусть бьет Бог тебя счастьем!’ Мэла́нка ходы́ла, Васы́лька просы́ла: «Васы́лько, мий ба́тько, Пусты́ мэнэ́ в ха́ту. Я жы́то нэ жа́ла, Пид сно́пом стоя́ла. Пид сно́пом стоя́ла, Золоты́й крэст дэржа́ла…» [VI]. Мэланка ходила, Василька просила: «Василька, мой батька, Пусти меня в дом, Я жита не жала, Под снопом стояла, Под снопом стояла, Золотой крест держала…» Мэла́нка ходы́ла, Мэланка ходила, Мэла́нка ходы́ла, Мэланка ходила, Василька просила: Васы́лька просы́ла: Васы́лька просы́ла: Василька просила: «Василька, мой батька, «Васы́лько, мий ба́тько, «Васы́лько, мий ба́тько, Пусты́ мэнэ́ в ха́ту. Пусти меня в дом, Пусты́ мэнэ́ в ха́ту. Я жы́то нэ жа́ла, Я жита не жала, Под снопом стояла, Пид сно́пом стоя́ла. Пид сно́пом стоя́ла, Под снопом стояла, Золотой крест держала…» В настоящем через несколько дней на со бранные ряжеными деньги снимают ресторан для своего круга. Мэланки должны завершить свой ход до полуночи, до того, как взрослые вместе с подростками начнут ходить посевать. Славянский обряд посевания в цыганской среде сохраняется в остаточной форме, потеряв пря мую связь с аграрной магией. По цыганским представлениям, ритуал обеспечивает хорошие заработки в наступающем году. р у В среде сохраняются представления о Чи стом четверге и Страстной (Страшной) пятнице. В Страстную пятницу особенно строго соблюда ется пост. Набожные цыгане ничего не едят до наступления темноты, после захода солнца могут съесть легкое постное блюдо. Считается, что в первый день Пасхи (Па тради́) (влахам и сэрвам) нельзя умываться, бриться, стричь ногти и т. д. Совершение этих гигиенических процедур воспринимается как неуважение к празднику. Неудача или несчастье, могущее постигнуть нарушителя, будет воспри нято окружением как наказание за нарушение этого запрета. Считается вежливым обязательно дождать ся посевальников. Они рассыпают по дому зер но, сыплют его хозяевам в ладони, на темя. Зерно могут смешивать с монетами. Его нельзя убирать три дня. Затем его сметают в угол или собирают и оставляют в стакане или другой емкости. Осенний цикл Прочие известные сэрвам и влахам празд ники, в том числе осеннего цикла, отмечаются походами в церковь, в основном пожилыми жен щинами. В такие дни не принято стирать и де лать уборку. В воскресенье или понедельник Фоминой недели отмечается поминальный день по умер шим родственникам – Проводкы́ (укр.) / Прóвод кэ (цыг.). В этот день посещают могилы, накры вают для умерших стол с обязательными блю дами: оставшимися с Пасхи варениками (влаш. каноррэ́), блинами с творогом (влаш. блы́нчикэ тиралэ́са) и сладким лапшевником (влаш. лапшэ́ныко) [5, c. 133]. Отношение к этому дню Весенний цикл Посе REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE 60 2020, Volumul XXVII E-ISSN: 2537-6152 у влахов и сэрвов кардинально разнится. Сэрвы воспринимают Проводкэ как праздник. Поздрав ляют друг друга, при встрече мужчины пожима ют руки. У влахов поздравлять с Проводками не принято, мужчины, встретившиеся в этот день на кладбище, избегают рукопожатий. На Пасху красят яйца и выпекают куличи (пáскэ, патрадя́). Куличи влахов и сэрвов могут достигать метра в высоту. С их изготовлением связана примета, что полая внутри пáска – пред знаменование смерти. Чтобы избежать несча стья, ее топят в реке [5. c. 133]. В ряде регионов Украины (Львов, Днепропе тровская обл.) сэрвы отмечают праздник Ка́ша (14 мая), в славянском календаре известный как Макарий, Еремей Запрягальник и т. д. Празд ник проводят, выезжая всей семьей на природу. Обязательное и основное блюдо этого дня – пше ничная каша, которую мужчины готовят на ко стре в чугунном казане12. По словам некоторых информантов, этот праздник отмечается только городскими сэрвами [V], и, видимо, в свое время был воспринят оседлыми цыганами у сельского окружения. Между тем информанты связывают Кашу со временами полуоседлости: «в это время пробовали землю: готова ли природа к кочевью, не сыро ли, что уже выросло в лесу и на лугах» [XII]. Однако данных, подтверждающих, что праздник отмечался кочующими цыганами, у нас нет. Еще одно центральное блюдо влашского пас хального стола – круглые вареники с мясом (ка норрэ́ ‘ушки’). Каноррэ́ не являются у влахов по вседневной едой, их готовят лишь дважды в год, на великие праздники: в период с Рождества до Крещения и с Пасхи до Вознесения (по́кы о Дэл пэ пхув, по́кы мо́жно тэ гэтóй каноррэ́ [VI] ‘пока Бог на земле, пока можно готовить каноррэ’. Каноррэ – поминальное блюдо, символизи рующее умерших членов семьи (братьев, сестер, дядей, теть и т. д.). Два самых больших – более трехсот граммов каждый – олицетворяют ба бушку и деда. Каноррэ носят по всей деревне (соседям, знакомым), предлагая помянуть усоп ших: «…идешь и свэнцомэ́ мас [освященное мясо], еще со-нибудь [что-нибудь], водку даже... несешь людям, чтоб помянули твоих померших!» [I]. Два последних оставляют себе и съедают под конец праздника: Каноррэ́ – о па́по й ба́ба, када́ со́ май-ғла́вни каноррэ́ а́ндай са́воррэ. Спэрва́ хас каноррэ́, ай со май-посли́дни, хас лэ папо́с, ни ка́дя пхэнэ́лпэ, ай тоди́ ла баба́ [III]. ‘Каноррэ – дед и бабушка, самые главные каноррэ из всех. Сперва ешь каноррэ, а самыми последними ешь деда, ну так говорится, а потом бабушку’. у Летний цикл К праздникам летнего цикла, отмечаемым влахами и сэрвами, относятся Троица (Трóй ца) и Спас (Спáсо). Троицу празднуют три дня, украшают дом (в прошлом – шатер) свежеско шенной травой и зелеными ветками неплодовых деревьев. Из трех Спасов (Яблочный, Медовый / Маковый и Ореховый) цыгане особо отмечают только первый (Преображение Господне). Мему арный источник конца XIX в. описывает в кон тексте украиноязычных неоседлых цыган Чер ниговской губернии праздник Ивана Купалы: «…тоже справляют праздник Ивана Купала. Дев ки и бабы кругом поют песню Купале, а мужчи ны и дети верхом на лошадях скачут через огонь, а когда огонь сделается меньше, то и девки» [2, с. 152]. В настоящем сэрвы считают этот празд ник нецыганским. За пасхальным столом влахи произносят: Сар арэсля́м кадá Патради́, бахтáса, лошáса, тэ встричáем джи кай шэл бэрш, май-лошалэ́с, инке́ пáла май-бахталэ́ скаминдá! [ЯП] ‘Как встретили эту Пасху, со счастьем, с радостью, чтоб встречали до ста лет, более радостно, еще за более счастливыми столами!’ Перед тостом произносящий его троекратно восклицает: Хры стос Воскрэс! Присутствующие отвечают ему хо ром. Универсальным тостом является: Тэ фэри́й тумэ́н о Дэл, кай тэ на жан, кай тэ на повэрнэ́т эсь, тэ шол о Дэл па́ла тумэ́ндэ бах! [VI] ‘Храни вас Бог, куда ни пойдете, где ни повернетесь, бро сает Бог за вами счастье!’ Примечания 7 Тэ андярэ́л вря́мя ‘нести время ’, тэ андярэ́л пэ́сти папо́сти вря́мя – нести время своего деда (жить по обычаям своего деда, придерживаться традицион ных устоев), ту ка́сти вря́мя андярэ́с? – чье время ты несешь? (по чьим традициям живешь?). Воз можно, в основе идиомы неточная калька с рус. нести бремя. XI. Зап. Я. А. Панченко от Ивана Андреевича Слиняв ского, 1939 г. р., таврического сэрва (род слыня́всь ки), г. Каховка Херсонской обл. Украины. XII. Зап Я. А. Панченко от Галины Николаевны Юр ченко, 1995 г. р., городской сэрвицы (род осэлэдци́), г. Львов, Украина. р 8 Это значит, что первый кусок надо съесть, стоя на топоре, а не на земле. у р у Заключение За описываемый период календарная обряд ность влахов и сэрвов, по всей видимости, не претерпела значительных изменений. С уверен ностью мы можем говорить о трансформации заимствованных цыганами обрядовых форм от носительно тех же обрядовых форм окружения. REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE 61 E-ISSN: 2537-6152 2020, Volumul XXVII Список информантов Мы отмечаем упрощение многих обрядов, утра ту аграрной символики и связи некоторых празд ников с культом православных святых. Перспек тива исследования связана с более подробным сравнением обрядности цыган и окружения, на селяющего регионы Украины и юга России. I. Зап. И. Ю. Махотиной от Татьяны Егоровны Ко нонюк, 1993 г. р., ставропольской власицы (род бэрэ́вуря). Родилась в г. Зеленокумск Советского р-на Ставропольского края. Прож. в Омске, РФ. II. Зап. Я. А. Панченко от Валентины Александровны Бардик (Орловой), нецыганки, 1963 г. р., г. Каховка Херсонской обл. Украины. Примечания III. Зап. Я. А. Панченко от Петра Вешановича Зайчен ко, 2000 г. р., ставропольского влаха (род мýрлуря), г. Зеленокумск Ставропольского края РФ. 1 Статья составлена И. Ю. Махотиной, Я. А. Панчен ко выступает в исследовании как основной соби ратель и один из информантов. В тексте коммен тарии Панченко отмечены инициалами ЯП. 1 Статья составлена И. Ю. Махотиной, Я. А. Панчен ко выступает в исследовании как основной соби ратель и один из информантов. В тексте коммен тарии Панченко отмечены инициалами ЯП. IV. Зап. Я. А. Панченко от Николая Николаевича Ма нуйлова, около сорока лет, таврического сэрва (род котони́), г. Ростов-на-Дону, РФ. 2 Исследование публикуется при частичной под держке Международного фонда «Возрождение» (Міжнародний фонд «Відродження») и REYN- Ukraine грант, № 52947. V. Зап. Я. А. Панченко от Маргариты Михайловны Мартиросян, 1965 г. р., по материнской линии го родская сэрвица (род флягы́ / хлягы́), г. Новая Ка ховка Херсонской обл. Украины. 3 Речь идет о ряде запретов, связанных с представ лениями о ритуальной нечистоте телесного низа (прежде всего замужней фертильной женщины, так как считается, что женщина более нечиста, чем мужчина; самые чистые – дети) – влаш. мағримóс, сэрв. мағримó. р р VI. Зап. Я. А. Панченко от родной бабушки по мате ринской линии Зинаиды Андреевны (Юренко) Миллер, (1948–2018), сэ́рвицы по отцу (род гу стэ́нкуря) и кубанской влáсицы по матери (род бýдкуря), г. Каховка Херсонской обл. Украины. р р 4 Название кочевые характеризует позднюю осед лость (после 1956 г.) этих групп относительно дру гих. VII. Зап. Я. А. Панченко от Владимира Николаенко, 1992 г. р., ставропольского влаха (род цокэ́нкуря), г. Ростов-на-Дону, РФ. 5 Предки современных цыган-влáхов переселились на Украину с территорий Валахии и Молдавии не ранее XVII в. Традиционно населяли придне стровские области Правобережной Украины, рас пространившись в результате миграций по всему ареалу проживания сэ́рвов – на юго-востоке Укра ины, в южных областях РФ [12, с. 14]. у VIII. Зап. Я. А. Панченко от Владимира Михайловича Панченко, 2000 г. р., таврического сэрва (род: пан ченкы́), г. Новая Каховка Херсонской обл. Украи ны. IX. Зап. Я. А. Панченко от двоюродной бабушки по отцовской линии Нелли Алексеевны Панченко, 1947 г. р., таврической сэрвицы (род панченкы́), г. Каховка Херсонской обл. Украины. Кочевала до 1956 г. 6 Вероятно, к волжским влахам относится зафикси рованное нами понятие вóложенские / валóжевские влахи. X. Зап. Я. А. Панченко от Петра Петровича Панченко, 1959 г. р., таврического сэрва (род панченкы́), г. 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Праздник Ка́ша (14 мая) у городских сэрвов из рода осэлэдци́, г. Львов, Украина, 2005 г. Приготовле ние главного блюда. Фото из архива Г. Н. Юрченко ( ) 1. Ряжения на Маланку у сэрвов рода панченкы́, г. Каховка Херсонской обл. Украины, январь, 2004 г. 4. Праздник Ка́ша (14 мая) у городских сэрвов из рода осэлэдци́, г. Львов, Украина, 2005 г. Приготовле ние главного блюда. Фото из архива Г. Н. Юрченко 1. Ряжения на Маланку у сэрвов рода панченкы́, г. Каховка Херсонской обл. Украины, январь, 2004 г. REVISTA DE ETNOLOGIE ȘI CULTUROLOGIE E-ISSN: 2537-6152 2020, Volumul XXVII 64 5. Рожэ́ствэнык, выпечка для лошадей у сэрвов-тав рычан из рода панченкы́, г. Каховка Херсонской обл. Украины, 5 января 2020 г. Фото из архива Я. А. Панченко Ilona Mahotina (Tver, Federația Rusă). Литература / References Doctor în fi lologie, Institutul de cercetare a țiganilor din Europa (Bel grad, Serbia). Ilona Mahotina (Tver, Federația Rusă). Doctor în fi lologie, Institutul de cercetare a țiganilor din Europa (Bel grad, Serbia). Илона Махотина (Тверь, Российская Федерация). Кандидат филологических наук, Институт исследова ний цыган Европы (Белград, Сербия). Ilona Makhotina (Tver, Russian Federation). PhD in Philology, Institute for the Roma studies of Europe (Bel grade, Serbia). E-mail: maxota@list.ru ORCID: https://orcid.org/0000-0003-0203-3124 Ilona Makhotina (Tver, Russian Federation). PhD in Philology, Institute for the Roma studies of Europe (Bel grade, Serbia). E-mail: maxota@list.ru E-mail: maxota@list.ru ORCID: https://orcid.org/0000-0003-0203-3124 Ianuș Pancenco. Doctorand, Universitatea Națională Zaporizhya (Ucraina). Януш Панченко Aспирант, Запорожский нацио нальный университет (Украина). Януш Панченко Aспирант, Запорожский нацио нальный университет (Украина). 5. Рожэ́ствэнык, выпечка для лошадей у сэрвов-тав рычан из рода панченкы́, г. Каховка Херсонской обл. Украины, 5 января 2020 г. Фото из архива Я. А. Панченко Yanush Panchenko PhD student, Zaporizhzhya Nati onal University (Ukraine). Yanush Panchenko PhD student, Zaporizhzhya Nati onal University (Ukraine). y E-mail: janushpanchenko@gmail.com ORCID: https://orcid.org/0000-0001-9974-3266
https://openalex.org/W3197545613	https://europepmc.org/articles/pmc8470870?pdf=render	English	null	Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modified Forms in Maize	Toxins	2,021	cc-by	7,668	Article Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modiﬁed Forms in Maize Marta Magdalena Sopel 1, Herbert Michlmayr 2 , Gerhard Adam 2 and Franz Berthiller 1,* Iris Fiby 1, Marta Magdalena Sopel 1, Herbert Michlmayr 2 , Gerhard Adam 2 and Franz 1 Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20, 3430 Tulln, Austria; iris.ﬁby@gmail.com (I.F.); marta_magdalena@onet.eu (M.M.S.) 1 Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20, 3430 Tulln, Austria; iris.ﬁby@gmail.com (I.F.); marta_magdalena@onet.eu (M.M.S.) 2 Institute of Microbial Genetics, Department of Applied Genetics and Cell Biology, BOKU, Konrad Lorenz Str. 24, 3430 Tulln, Austria; herbert.michlmayr@boku.ac.at (H.M.); h d d @b k (G A ) 1 Institute of Bioanalytics and Agro-Metabolomics, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz Str. 20, 3430 Tulln, Austria; iris.ﬁby@gmail.com (I.F.); marta_magdalena@onet.eu (M.M.S.) 2 , ; y g ( ); g ( ) 2 Institute of Microbial Genetics, Department of Applied Genetics and Cell Biology, BOKU, Konrad Lorenz Str. 24, 3430 Tulln, Austria; herbert.michlmayr@boku.ac.at (H.M.); gerhard.adam@boku.ac.at (G.A.) y g g 2 Institute of Microbial Genetics, Department of Applied Genetics and Cell Biology, BOKU, Konrad Lorenz Str. 24, 3430 Tulln, Austria; herbert.michlmayr@boku.ac.at (H.M.); gerhard.adam@boku.ac.at (G.A.) g * Correspondence: franz.berthiller@boku.ac.at; Tel.: +43-1-47654-97371 * Correspondence: franz.berthiller@boku.ac.at; Tel.: +43-1-47654-97371 Abstract: The Fusarium mycotoxin deoxynivalenol (DON) is a common contaminant of cereals and is often co-occurring with its modiﬁed forms DON-3-glucoside (D3G), 3-acetyl-DON (3ADON) or 15- acetyl-DON (15ADON). A stable-isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) based method for their determination in cereals was developed and validated for maize. Therefore, 13C-labelled D3G was enzymatically produced using 13C-DON and [13C6Glc]- sucrose and used as an internal standard (IS) for D3G, while uniformly 13C labelled IS was used for the other mycotoxins. Baseline separation was achieved for the critical peak pair DON/D3G, while 3ADON/15ADON could not be fully baseline separated after testing various reversed phase, ﬂuorinated phase and chiral LC columns. After grinding, weighing and extracting the cereal samples, the raw extract was centrifuged and a mixture of the four 13C-labelled ISs was added directly in a microinsert vial. The subsequent analytical run took 7 min, followed by negative electrospray ionization and selected reaction monitoring on a triple quadrupole MS. Maize was used as a complex cereal model matrix for validation. Citation: Fiby, I.; Sopel, M.M.; Michlmayr, H.; Adam, G.; Berthiller, F. Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modiﬁed Forms in Maize. Toxins 2021, 13, 600. https://doi.org/ 10.3390/toxins13090600 Keywords: mycotoxins; trichothecenes; masked mycotoxins; modiﬁed mycotoxins; mass spectrome- try; stable-isotope dilution assay Received: 28 June 2021 Accepted: 25 August 2021 Published: 27 August 2021 Key Contribution: A novel highly accurate liquid chromatography-tandem mass spectrometry method for the determination of deoxynivalenol and its modiﬁed forms was developed and validated for maize. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Article Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modiﬁed Forms in Maize The use of the IS corrected the occurring matrix effects efﬁciently from 76 to 98% for D3G, from 86 to 103% for DON, from 68 to 100% for 15ADON and from 63 to 96% for 3ADON. Citation: Fiby, I.; Sopel, M.M.; Michlmayr, H.; Adam, G.; Berthiller, F. Development and Validation of an LC-MS/MS Based Method for the Determination of Deoxynivalenol and Its Modiﬁed Forms in Maize. Toxins 2021, 13, 600. https://doi.org/ 10.3390/toxins13090600 toxins toxins toxins toxins 1. Introduction DON is often co-occurring with its acetylated biosynthetical precursors 3-acetyl-DON 3ADON) or 15-acetyl-DON (15ADON), and its plant metabolite DON-3-glucoside (D3G, ) or 15-acetyl-DON (15ADON), and its plant metabolite DON-3-glucoside ) in cereals, such as wheat, barley, oats, rye and maize or products thereof (a) (d) (c) (b) Figure 1. Chemical structures of (a) deoxynivalenol (DON) and its modified forms (b) deoxyniva lenol-3-glucoside (D3G) (c) 3-acetyldeoxynivalenol (3ADON) and (d) 15-acetyldeoxynivaleno Figure 1. Chemical structures of (a) deoxynivalenol (DON) and its modiﬁed forms (b) deoxynivalenol-3-glucoside (D3G), (c) 3-acetyldeoxynivalenol (3ADON) and (d) 15-acetyldeoxynivalenol (15ADON). (a) (b) (a) (b) (a) (b) (d) (c) (c) Figure 1. Chemical structures of (a) deoxynivalenol (DON) and its modified forms (b) deoxyniv lenol-3-glucoside (D3G) (c) 3-acetyldeoxynivalenol (3ADON) and (d) 15-acetyldeoxynivalen Figure 1. Chemical structures of (a) deoxynivalenol (DON) and its modiﬁed forms (b) deoxynivalenol-3-glucoside (D3G), (c) 3-acetyldeoxynivalenol (3ADON) and (d) 15-acetyldeoxynivalenol (15ADON). ( ), ( ) y y ( ) ( ) y y t popular technique to determine mycotoxins in food nowadays [11,12] is atography coupled to mass spectrometry (LC-MS). Typically reversed phase phy on a C-18 column is used to separate mycotoxins of different polarities, re charged during electrospray ionization and subsequently analyzed with MS offers tremendous sensitivity, selectivity and multiplexing capability, but ntification is often challenged due to matrix effects [14]. As such, co-eluting ounds suppress or enhance the signal (SSE), compared to standards in neat rnal calibration). One of the most sophisticated ways to cope with matrix sage of stable isotope labeled internal standards. Those have the same phys- properties as the analytes, but different molecular masses and do not occur eral stable isotope dilution assays (SIDA) have been developed for accurate termination so far, including the most important mycotoxins (e.g., [15–17]). The most popular technique to determine mycotoxins in food nowadays [11,12] is liquid chromatography coupled to mass spectrometry (LC-MS). Typically reversed phase chromatography on a C-18 column is used to separate mycotoxins of different polarities, before they are charged during electrospray ionization and subsequently analyzed with MS [13]. LC-MS offers tremendous sensitivity, selectivity and multiplexing capability, but accurate quantiﬁcation is often challenged due to matrix effects [14]. As such, co- eluting matrix compounds suppress or enhance the signal (SSE), compared to standards in neat solvents (external calibration). One of the most sophisticated ways to cope with matrix effects is the usage of stable isotope labeled internal standards. 1. Introduction Mycotoxins are low molecular weight, secondary metabolites of fungi of different gen- era, which may cause serious health implications for mammals, when ingested with food or feed, as reviewed by [1]. One of the most prevalent groups of mycotoxins—trichothecenes— contains a tetracyclic sesquiterpenoid 12,13-epoxytrichothec-9-en ring structure, with the epoxide group responsible for the typical trichothecenes’ toxic effects, as reviewed by [2]. Trichothecenes are produced by plant pathogenic Fusarium spp., growing preferably on cereals in the ﬁeld at temperate climates [3]. The type B trichothecene deoxynivalenol (DON) is one of the most commonly found mycotoxins worldwide [4]. Its toxic effects include emesis (hence, its colloquial name “vomitoxin”), anorexia, growth retardation, im- munotoxicity, impaired reproduction and development, altered neuroendocrine signaling, proinﬂammatory gene induction and altered gut integrity [5]. 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/toxins Toxins 2021, 13, 600. https://doi.org/10.3390/toxins13090600 Toxins 2021, 13, 600 doc 2 of 12 t l DON is often co-occurring with its acetylated biosynthetical precursors 3-acetyl-DON (3ADON) or 15-acetyl-DON (15ADON), and its plant metabolite DON-3-glucoside (D3G, Figure 1) in cereals, such as wheat, barley, oats, rye and maize or products thereof (e.g., [6,7]). As both the acetylated and the glucosidic forms of DON can be easily hydrolyzed to DON in vivo [8], the toxicity of those so-called “modiﬁed mycotoxins” [9] is basically the same as that of the DON for humans. As such, the European Food Safety Authority proposed a group tolerable daily intake value of 1 µg/kg bodyweight for the sum of the four compounds [10]. N) or 15-acetyl-DON (15ADON), and its plant metabolite DON-3-glucoside 1) in cereals, such as wheat, barley, oats, rye and maize or products thereof s both the acetylated and the glucosidic forms of DON can be easily hydro- N in vivo [8], the toxicity of those so-called “modified mycotoxins” [9] is ba- me as that of the DON for humans. As such, the European Food Safety Au- sed a group tolerable daily intake value of 1 µg/kg bodyweight for the sum mpounds [10]. 1. Introduction Those have the same physico-chemical properties as the analytes, but different molecular masses and do not occur in nature. Several stable isotope dilution assays (SIDA) have been developed for accurate mycotoxin determination so far, including the most important mycotoxins (e.g., [15–17]). While uniformly labeled 13C-DON, 13C-3ADON and 13C-15ADON are commercially available, until very recently, 13C-D3G was not. Habler et al. proposed the Königs–Knorr method to chemically synthesize DON-3-[13C6]-glucoside from unlabeled DON and [13C6]-labeled glucose. The authors successfully applied the compound as IS for the analysis of beer samples for the concurrent determination of DON and D3G [18]. mly labeled 13C-DON, 13C-3ADON and 13C-15ADON are commercially avail- ry recently, 13C-D3G was not. Habler et al. proposed the Königs–Knorr emically synthesize DON-3-[13C6]-glucoside from unlabeled DON and [13C6]- se. The authors successfully applied the compound as IS for the analysis of f th t d t i ti f DON d D3G [18] Another challenge in mass spectrometry is the determination of isomers, as they—by deﬁnition—share the same molecular formula and mass. If speciﬁc MS/MS fragmentation is unavailable, chromatographic separation should be aimed for. In the case of 3ADON and 15ADON, this separation is hard to achieve and in many multi-toxin methods, those two compounds co-elute (e.g., [19]). While the loss of a CH2O group at C-15 during collision Toxins 2021, 13, 600 3 of 12 induced dissociation in MS/MS allows the formation of a speciﬁc fragment of 3ADON in negative ion mode (m/z 307), no such speciﬁc ion is available for 15ADON, severely limiting its quantiﬁcation. g q The major aim of this work was to develop and validate a robust, fast and accurate LC-MS/MS based method that allows the concurrent determination of DON along with its major modiﬁed forms 3ADON, 15ADON and D3G. We enzymatically produced and puriﬁed uniformly labeled 13C-D3G and used it together with 13C-DON, 13C-3ADON and 13C-15ADON as internal standards. Chromatographic separation was optimized, allowing near base-line separation of 3ADON and 15ADON. To the best of our knowledge, this is the ﬁrst stable-isotope dilution assay covering those four mycotoxins. 2.1. MS Method Optimization One of the main prerequisites to developing a SIDA method was the production of 13C-D3G, which was commercialized later. U-[13C21]-D3G was synthesized in a batch conversion containing 100 mg 13C-DON. The batch contained 21 mM U-[13C15]-DON, 32 mM [13C6Glc]-sucrose (β-D-fructofuranosyl-α-D-[U-13C6]glucopyranoside; 99 atom% 13C, Omicron Biochemicals Inc., South Bend, IN, USA), 1 mM UDP, 100 mM potassium phosphate pH 7. UDP-glucosyltransferase OsUGT79 and sucrose synthase AtSUS1 [20] were added at 1.5 mg/mL each. The reaction was incubated at 37 ◦C. After 48 hrs, the batch contained less than 0.5% un-conjugated DON. U-[13C21]-D3G was isolated by preparative HPLC (Agilent 1100 series, Waldbronn, Germany) and freeze-dried. The total yield was 76%. The electrospray (ESI) MS/MS fragmentation spectra of the produced compound, as well as those of unlabeled D3G at 30 eV collision energy, are shown in Figure 2. All major fragments of D3G were found with 13C-D3G with the according mass shifts (+21 amu for the precursor due the sum formula of C21H30O11). One of the main fragments is the loss of a CH2O group from the C-15 backbone of DON (m/z 427 or 447 for the labeled compound). Despite the same collision energy, that fragment was more abundant than the deprotonated precursor of 13C-D3G, but less abundant using the deprotonated D3G as precursor for reasons unknown. However, the acetate adduct resulted in much higher overall intensities and the fragmentation pattern of D3G and of 13C-D3G were virtually identical. Consecutive syringe pump optimization was performed for all analytes and IS for both the acetate adducts and the deprotonated precursors and used for method development. After coupling with LC, it could be seen that the acetate adducts of all analytes gave higher signal to noise ratios; hence, three transitions for each analyte—always using the [M+CH3COO]−precursors—were selected for the ﬁnal method (Table 1). The entrance potentials were kept at 10 V for each transition. Dwell times of 20 ms and pause times of 5 ms between transitions resulted in a cycle time of 0.45 s. 4 of 13 Figure 2. Cont. Figure 2. Cont. 4 of 12 Toxins 2021, 13, 600 Figure 2. Enhanced Product Ion (MS/MS) spectra of D3G (left) and 13C-D3G (right) at a collision energy of 30 eV. The spectra derived from the acetate adducts are shown on top, the spectra using Figure 2. Enhanced Product Ion (MS/MS) spectra of D3G (left) and 13C-D3G (right) at a collision energy of 30 eV. 2.1. MS Method Optimization The spectra derived from the acetate adducts are shown on top, the spectra using the deprotonated precursors on the bottom. Figure 2. Enhanced Product Ion (MS/MS) spectra of D3G (left) and 13C-D3G (right) at a collision energy of 30 eV The spectra derived from the acetate adducts are shown on top the spectra using Figure 2. Enhanced Product Ion (MS/MS) spectra of D3G (left) and 13C-D3G (right) at a collision energy of 30 eV. The spectra derived from the acetate adducts are shown on top, the spectra using the deprotonated precursors on the bottom. p precursors on the bottom. Table 1. List of analytes with optimized ESI-MS/MS parameters. precursors on the bottom. e syringe pump optimization was performed for all analytes and IS for adducts and the deprotonated precursors and used for method develop- pling with LC, it could be seen that the acetate adducts of all analytes gave noise ratios; hence, three transitions for each analyte—always using the precursors—were selected for the final method (Table 1). The entrance po- pt at 10 V for each transition. Dwell times of 20 ms and pause times of 5 nsitions resulted in a cycle time of 0.45 s. Table 1. List of analytes with optimized ESI-MS/MS parameters. Analyte ID Q1 Mass (Da) Q3 Mass (Da) DP (V) CE (eV) CXP (V) D3G 1 517.1 457.1 −80 −22 −7 D3G IS 1 538.1 478.1 −80 −22 −7 D3G 2 517.1 59.0 −80 −74 −9 D3G IS 2 538.1 59.0 −80 −74 −9 D3G 3 517.1 427.0 −80 −32 −5 D3G IS 3 538.1 447.0 −80 −32 −5 DON 1 355.0 59.0 −70 −36 −9 DON IS 1 370.0 59.0 −70 −36 −9 DON 2 355.0 295.0 −70 −16 −13 DON IS 2 370.0 310.0 −70 −16 −13 DON 3 355.0 265.0 −70 −24 −13 DON IS 3 370.0 279.0 −70 −24 −13 ADONs 1 397.1 59.0 −70 −34 −9 ADON IS 1 414.1 49.0 −70 −34 −9 ADONs 2 397.1 337.1 −70 −12 −7 ADON IS 2 414.1 354.1 −70 −12 −7 3ADON 397.1 307.0 −70 −22 −5 3ADON IS 414.1 323.0 −70 −22 −5 D3G deoxynivalenol-3-glucoside, DON deoxynivalenol, ADONs acetyl-deoxynivalenols, 3ADON 3-acetyl- deoxynivalenol, IS internal standard, DP declustering potential, CE collision energy, CXP cell exit potential. 2.2. LC Method Optimization Different types of chromatography (reversed phased, chiral) and 13 different columns (including C18 and pentaﬂuorophenyl phases) were evaluated to separate the critical peak pairs of DON/D3G and 3ADON/15ADON. Ammonium acetate (2 mM) was added into both mobile phases to ensure acetate ion adduct formation in negative ESI mode. With all conditions, acetonitrile (ACN) gave better separation for 3ADON/15ADON than methanol (MeOH) and was therefore chosen as the organic solvent for the ﬁnal method. For DON/D3G, separation was similar for both mobile phases. The retention times and resolutions of the critical peak pairs for the tested columns, using the ACN mobile phase, are summarized in Table 2. Toxins 2021, 13, 600 5 of 12 Table 2. Evaluated analytical columns for the LC-MS/MS method. Column tR (DON) (min) tR (D3G) (min) tR (3ADON) (min) tR (15ADON) (min) FWHM (min) RS (DON/D3G) RS (3/15ADON) Agilent Zorbax Eclipse Plus 2.63 2.68 4.16 4.17 0.045 −1.11 −0.22 Agilent Zorbax Extend-C18 2.15 2.26 3.85 3.86 0.077 −1.43 −0.13 Agilent Poroshell EC-C18 5.30 5.14 7.30 7.16 0.057 2.82 2.47 Agilent Zorbax SB C18 5.87 5.87 9.00 9.02 0.085 0.00 −0.24 Agilent Zorbax XDB-C18 4.75 4.52 6.58 6.46 0.060 3.83 2.00 Daicel Chiralcel 3.59 3.45 11.00 9.00 3.000 0.05 0.67 Daicel Chiralpak 2.29 2.06 9.29 9.08 0.143 1.60 1.47 Phenomenex Kinetex C18 4.07 4.03 5.81 5.82 0.075 0.53 −0.13 Phenomenex Kinetex F5 4.26 4.15 6.33 6.23 0.067 1.65 1.50 Sigma Discovery HS F5 6.14 5.95 9.88 9.68 0.122 1.56 1.64 Thermo Hypersil Gold 1.84 2.13 3.68 3.69 0.112 −2.60 −0.09 Waters Acquity BEH C18 2.98 3.21 4.37 4.40 0.040 −5.75 −0.75 Waters Acquity HSS T3 C18 5.20 4.93 7.17 7.02 0.052 5.23 2.90 tR retention time, FWHM full width half maximum, Rs resolution. A water–acetonitrile gradient (5% →60% acetonitrile in 9 min) containing 2 mM ammonium acetate was used for separation. The ﬂow rate was 0.2 or 0.4 mL/min, depending on the column length. Table 2. Evaluated analytical columns for the LC-MS/MS method. tR retention time, FWHM full width half maximum, Rs resolution. A water–acetonitrile gradient (5% →60% acetonitrile in 9 min) containing 2 mM ammonium acetate was used for separation. The ﬂow rate was 0.2 or 0.4 mL/min, depending on the column length. Only three columns (marked bold in Table 2) yielded resolutions ≥2.0 for both peak pairs. Of those, the Waters Acquity HSS T3 yielded the highest separation power and was used for further optimization. 2.2. LC Method Optimization The gradient was optimized and the run time shortened to develop the ﬁnal method (see chromatogram in Figure 3). 2.3. Method Validation Separation was achieved on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column using a water–acetonitrile gradient. Figure 3. LC-MS/MS selected ion monitoring (SRM) chromatogram of the optimized method. Sep- aration was achieved on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column using a Figure 3. LC-MS/MS selected ion monitoring (SRM) chromatogram of the optimized method. Separation was achieved on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column using a water–acetonitrile gradient. water–acetonitrile gradient. Table 3. Method performance parameters. . Table 3. Method performance parameters. water–acetonitrile gradient. 2.3. Method Validation Prior to method validation, different extraction solvents (20%, 50%, 80% aqueous ACN) were tested. All three solvents yielded almost identical apparent recoveries (RA). As expected, the matrix effects (SSE) were slightly less pronounced with more apolar sol- vents, while the extraction recoveries (RE) were slightly higher with more polar solvents Table 3. Method performance parameters. Analyte LOQ Solution (µg/L) LOQ Maize (µg/kg) RE (%) SSE (%) RA (%) RSDr (%) D3G (ext.) <10 <40 94.7 75.9 92.3 3.1 D3G (int.) 97.5 8.3 DON (ext.) <10 <40 101 86.4 104 5.9 DON (int.) 103 5.3 15ADON (ext.) <30 <120 105 67.8 105 9.4 15ADON (int.) 100 7.8 3ADON (ext.) <10 <40 94.4 63.2 90.8 7.0 3ADON (int.) 96.2 5.7 LOQ: limit of quantiﬁcation; RE: extraction recovery; SSE: signal suppression or enhancement; RA: apparent recovery; RSDr: relative standard deviation under conditions of repeatability; ext. external calibration; int. internal calibration. LOQ: limit of quantiﬁcation; RE: extraction recovery; SSE: signal suppression or enhancement; RA: apparent recovery; RSDr: relative standard deviation under conditions of repeatability; ext. external calibration; int. internal calibration. 2.3. Method Validation Prior to method validation, different extraction solvents (20%, 50%, 80% aqueous ACN) were tested. All three solvents yielded almost identical apparent recoveries (RA). As expected, the matrix effects (SSE) were slightly less pronounced with more apolar solvents, while the extraction recoveries (RE) were slightly higher with more polar solvents (data not shown). As the matrix effects are supposed to be corrected by the IS, ACN/H2O, 20/80 (v/v) was chosen as extraction solvent. In addition, this yielded the advantage that the raw solvent can be directly injected into the LC-MS/MS system without causing peak distortion. The determined method performance parameters for maize include apparent recovery (RA), matrix effects (SSE) and extraction recovery (RE) and are summarized in Table 3. For limits of quantiﬁcation (LOQ) determination, the spiked maize samples were used which consistently yielded signal to noise ratios exceeding 10. Linearity has been shown for neat standard solutions in the range of 3–1000 µg/L (equaling 12–4000 µg/kg) with squared linear calibration coefﬁcients exceeding 0.998 for all analytes. 6 of 12 Toxins 2021, 13, 600pa develop the final method (see chromatogram in Figure 3). Figure 3. LC-MS/MS selected ion monitoring (SRM) chromatogram of the optimized method. Sep- aration was achieved on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column using a 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 time [min] 0.0 0.4 0.8 1.2 1.6 2.0 2.4 Intensity [105 cps] D3G 1.70 min DON 1.95 min 15ADON 3.23 min 3ADON 3.31 min Figure 3. LC-MS/MS selected ion monitoring (SRM) chromatogram of the optimized method. Separation was achieved on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column using a water–acetonitrile gradient. develop the final method (see chromatogram in Figure 3). 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 time [min] 0.0 0.4 0.8 1.2 1.6 2.0 2.4 Intensity [105 cps] D3G 1.70 min DON 1.95 min 15ADON 3.23 min 3ADON 3.31 min Figure 3. LC-MS/MS selected ion monitoring (SRM) chromatogram of the optimized method. Sep- aration was achieved on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column using a Figure 3. LC-MS/MS selected ion monitoring (SRM) chromatogram of the optimized method. 3. Discussion Several different extraction solvents were tested in this study. In (multi-analyte) mycotoxin determination, a very common solvent is ACN/H2O/acetic acid (79/20/1, v/v/v) [19]. Acidiﬁcation is important for the extraction of several (charged) mycotoxins, but not for type B-trichothecenes, e.g., [21,22]. The high ratio of organic solvent usually serves multiple purposes: (a) more apolar mycotoxins than type B-trichothecenes can be extracted, (b) the solvent is compatible with several clean-up strategies and (c) fewer polar matrix compounds are co-extracted, yielding fewer matrix effects for polar analytes. Considering compensation of matrix effects by the used ISs, we opted for the solvent composition with the highest extraction recoveries, which was ACN/H2O, 20/80 (v/v). Choosing this solvent also allows direct injection in the UHPLC system after the addition of ISs and avoids a further dry-down step. y p More sensitive methods for the determination of type B-trichothecenes are available in literature, recently reviewed in [23]. Such methods often need a clean-up and concentration step, which could be avoided here. Sample preparation is not only faster and cheaper (even considering the costs of the ISs), but less error-prone and more robust. This was made possible by the use of a highly sensitive MS system. In case such a system is unavailable, up-concentration is a requirement to achieve suitable limits of quantiﬁcation. Mass spectrometric performance varies from day to day, and in the worst case, also during measurement. The use of standards before, after and in the middle of the sequence is therefore recommended. We did notice only a negligible decrease of the slopes of the individual calibration curves during a batch, allowing us to use all replicates for the evaluation of the results. We did, however, notice day-to-day variations in sensitivity, as the instrument is used by multiple users and running different methods (and matrices). As such, we refrained from the determination of limits of detections, which is often performed under optimal system performance. Instead, we used standards with concentrations of 3, 10, 30, 100 µg/L and deﬁned the LOQ for that level which always showed signal/noise ratios higher than 10. This approach resulted in LOQs of 10 µg/L (40 µg/kg) for all analytes, but the rather poorly ionizing 15ADON (LOQ 30 µg/L or 120 µg/kg). With maximum regulated levels of 200–1750 µg/kg for DON in Europe [24], we consider the method sensitive enough for routine applications. 3. Discussion Our aim was to develop a robust yet accurate, fast and easy to use LC-MS/MS based method for the determination of DON and its major metabolites for routine food analysis. In order to do so, it was ﬁrst imperative to produce and characterize 13C-D3G, which is now available to all stakeholders. The applied enzymatic strategy [20] can be easily scaled up and yielded uniformly labelled 13C21-D3G for the ﬁrst time, already using 13C15-DON [15] as starting compound. g p Another—often ignored—issue in mycotoxin determination was partly solved with the accurate quantiﬁcation of 3ADON and 15ADON. The two compounds can be easily separated using gas chromatography [21], but barely using LC even by applying long run-times [22]. Fusarium spp. originally produce 3,15-diacetyl-DON that in the ﬁnal step of biosynthesis is deacetylated by an esterase encoded by the TRI8 gene [2]. Different alleles of this gene determine production of either 3ADON or 15ADON chemotypes in F. graminearum. While often samples are only contaminated with one chemotype, e.g., com- Toxins 2021, 13, 600 7 of 12 plex food samples or mixed feedstuffs easily can contain both acetylated forms. For a toxicological point of view, it is important to differ between the compounds, as there are some differences in gastro-intestinal deleterious effects and relative toxicity, with 15ADON often being the more potent toxin [8]. From an analytical-chemical point of view, it is relevant to observe differences in ionization of the compounds (see Figure 3), so a sum value with either or both toxins, as standards will lead to inaccurate quantiﬁcation when there is no chromatographical separation. Multi-mycotoxin methods based on LC-MS/MS are widely used nowadays [11,12], but remain a compromise in various aspects in order to “squeeze in” a multitude of analytes in a single method. In case accurate quantiﬁcation of 3ADON and 15ADON is warranted, methods not resolving the compounds cannot be recommended. Due to the speciﬁc fragmentation of 3ADON, this compound can be accurately determined even in the presence of 15ADON with such methods, e.g., [19]. While this proposed method does not fully solve the issue, the separation of 3ADON and 15ADON is sufﬁcient to accurately quantify them. While we used the peak areas for the quantiﬁcation (due to only low crosstalk from the overlapping peaks), a suitable option would be to use the peak heights instead (after using the same peak smoothing settings for all samples and standards). 4.2. Samples Different maize samples (1 kg each) used for the method development were bought at a health food store in Tulln, Austria, and milled with a Romer Analytical Sampling Mill from Romer Labs GmbH (Getzersdorf, Austria). For the determination of natural mycotoxin contamination, samples were extracted and measured with a multi-mycotoxin method [19]. A popcorn maize from controlled organic cultivation (Rapunzel Naturkost, Germany) was shown to be uncontaminated with 3ADON, 15ADON and D3G and was used as a blank for method validation. DON was only found in traces, below the LOQ of the used method (<10 µg/kg). 4.1. Chemicals Acetonitrile (ACN, gradient grade) was purchased from VWR International GmbH (Vienna, Austria), methanol (MeOH, ≥99.9 %) was obtained from Honeywell (Seelze, Germany), ammonium acetate (LC-MS grade) was provided by Sigma Aldrich (Vienna, Austria). Ultrapure water was produced by an ELGA Purelab Ultra system (Celle, Ger- many). All standards were provided by Romer Labs GmbH (Tulln, Austria). The in- dividual stock standard solutions (all in ACN) had the following concentrations: DON 100.5 µg/mL, 3ADON 100.4 µg/mL, 15ADON 100.1 µg/mL, D3G 50.4 µg/mL, U-[13C15]- DON 25.1 µg/mL (99.0 atom% 13C), U-[13C17]-3ADON 25.2 µg/mL (99.4 atom% 13C), U-[13C17]-15ADON 10.0 µg/mL (99.1% 13C). U-[13C21]-D3G became recently available from Romer Labs (10.6 µg/mL, 99.2 atom% 13C). 3. Discussion In case low background levels are the study subject, an intermediate up-concentration step would be required. On purpose, we added the ISs only after extraction, only compensating matrix effects during measurements and random injection volume variations. The use of conical micro- inserts allows minimizing the amount of ISs, thus saving costs. Unconventionally, we also opted to simply dilute 80 µL of raw extracts with 20 µL of ISs in the vials—resulting in a dilution factor for the samples. However, this factor is offset if the standards are prepared in the same manner. For example, 80 µL of a 100 µg/L neat standard solution Toxins 2021, 13, 600 8 of 12 are diluted with 20 µL of ISs, but further regarded as 100 µg/L standard (despite its actual concentration of only 80 µg/L). Using this little trick, no back calculations (other than the dilution factor of four for extraction) are needed for data evaluation. It is sometimes believed that the repeatability of a method could always be improved using ISs. In our case, which is in agreement with current literature, e.g., [16,17], this was not the case. Already excellent RSDr values of 3–9% for all analytes using external calibra- tion changed to 5–8% using internal calibration. Expected interlaboratory reproducibilies RSDR of 32.0%, 22.6% and 16.0% for levels of 10, 100 and 1000 µg/kg, according to [25], are likely to be reached. Thus, even further improvement of the method precision by using IS was unlikely and not obtained. Concluding, the presented method offers a robust manner to accurately determine DON and its major metabolites in cereals, using minimal sample preparation if a highly sensitive LC-MS/MS system is available. 4.3. LC-MS/MS Optimization All LC-MS/MS measurements were performed on a 1290 series ultra-high perfor- mance liquid chromatography system (Agilent Technologies, Waldbronn, Germany) cou- pled to a QTrap 6500+ MS/MS System (Sciex, Foster City, CA, USA) equipped with a IonDrive Turbo V electrospray ionization (ESI) source. Analysis was carried out using the dynamic selective reaction monitoring mode (SRM) with monitoring of two transitions (quantiﬁer and qualiﬁer). Precursor and product ion selection as well as the optimization of declustering poten- tials (DP), entrance potentials (EP), collision energies (CE) and cell exit potentials (CXP) were performed with ﬂow injection of single analyte solutions of 1 mg/L concentration us- ing a Hamilton syringe and the Analyst 1.6.3. software in negative mode (ESI-). The source temperature was 550 ◦C. From each analyte, the acetate adduct and the deprotonated adduct were scanned. For the optimization of the separation of DON, D3G, 3ADON and 15ADON, a 1 mg/L working solution in ACN/H2O, 40/60, v/v was used. Different UHPLC columns, of C18, perﬂuorinated and chiral materials were tested for this purpose using two following LC methods, where eluent A was composed of 5% MeOH (or ACN) and eluent B of 98% Toxins 2021, 13, 600 9 of 12 9 of 12 MeOH (or ACN), both containing 2 mM ammonium acetate. Chromatographic separation was performed at 25 ◦C with a ﬂow rate of 0.4 mL/min for columns with a length of 50 mm, and a ﬂow rate of 0.2 mL/min for columns with a length of 100 or 150 mm. The injection volume was set to 3 µL. The total chromatographic run time was 15 min. Different gradients’ initial conditions (10, 20 and 30% of B) and slopes with an intermediate step at (20, 30, 40 and 50% B) were tested prior to shortening the method from 15 to 7 min. Tested columns were purchased from Agilent Technologies (Waldbronn, Germany), Daicel (Chiral Technologies Europe SAS, Illkirch-Graffenstaden, France), Phenomenex (Aschaffenburg, Germany), Sigma-Aldrich, ThermoFisher Scientiﬁc (Vienna, Austria) or Waters (Vienna, Austria) and are shown in Table 4. Table 4. Evaluated analytical columns for the LC-MS/MS method. 4.4. Method Validation Recovery experiments were performed by spiking blank maize samples (1.00 ± 0.01 g) with the appropriate amount of spiking solution of unlabeled mycotoxins at six levels (resulting in expected measurement values of 3, 10, 30, 100, 300 and 1000 µg/L) in triplicate before extraction. The concentrations of spiking solutions used were 10.0 mg/L, 1.00 mg/L and 100 µg/L of DON, D3G, 3ADON and 15ADON solved in pure ACN. Fifteen mL polypropylene tubes with spiked samples were allowed to rest in the hood overnight at room temperature to allow solvent evaporation and to achieve equilibrium between the analytes and matrix. On the next day, the samples were extracted with 4.00 mL ACN/H2O, 20/80 (v/v) for 60 min on a shaker at room temperature and centrifuged (3500 rpm). Subsequently, 20 µL of IS working solution consisting of a 500 µg/L concentration of 13C-DON, 13C-D3G, 13C-3ADON and 13C-15ADON dissolved in ACN/H2O, 20:80, v/v, was added to 80 µL of the supernatant in an HPLC vial ﬁtted with a 200 µL conical glass insert. To evaluate matrix effects, blank maize samples (5.00 ± 0.01 g) were extracted in triplicates with 20 mL of ACN/H2O, 20/80 (v/v) for 60 min on a rotary shaker (200 rpm) at room temperature and centrifuged (3500 rpm). Matrix-matched standards were prepared at six levels (+a blank level) in triplicates. For this purpose, working standard solutions of mycotoxins were pipetted into HPLC vials, evaporated and reconstituted with 500 µL of raw extract. This resulted in a spiking level of 0, 3.00, 10.0, 30.0, 100, 300 and 1000 µg/L. Afterwards, 80 µL of these solutions were mixed with 20 µL IS working solution in an HPLC vial containing a microinsert. g Sample preparation in general consisted of cereal extraction with the four-fold volume of ACN/H2O, 20/80 (v/v) for 60 min on a rotary shaker, followed by centrifugation. Always, 80 µL of either raw extracts or standard solutions in neat solvents were diluted with 20 µL IS solution (500 µg/L of all four labelled compounds) directly in the HPLC microinsert prior to analysis. 4.3. LC-MS/MS Optimization Supplier Brand Name Dimensions (mm) Particle Size (µm) Agilent ZORBAX RRHD Eclipse Plus C18 2.1 × 50 1.8 Agilent ZORBAX RRHT Extend-C18 2.1 × 50 1.8 Thermo Hypersil GOLD C18 2.1 × 50 1.9 Waters ACQUITY UPLC BEH C18 2.1 × 50 1.7 Agilent ZORBAX RRHD StableBond C18 2.1 × 100 1.8 Agilent ZORBAX RRHD Eclipse XDB-C18 2.1 × 100 1.8 Phenomenex Kinetex C18 2.1 × 100 2.6 Waters ACQUITY UPLC HSS T3 (C18) 2.1 × 100 1.8 Agilent InﬁnityLab Poroshell 120 EC-C18 2.1 × 150 2.7 Phenomenex Kinetex F5 2.1 × 100 2.6 Sigma- Aldrich Discovery HS F5 2.1 × 100 5.0 Daicel CHIRALPAK AD-3R 2.1 × 150 3.0 Daicel CHIRALCEL OJ-3R 2.1 × 150 3.0 Table 4. Evaluated analytical columns for the LC-MS/MS method. As DON/D3G as well as 3ADON/15ADON yielded nearly identical peak width, the resolution was calculated with the following equation. RS = t2 −t1 w (1) (1) Unconventionally, we used the peak at the half maximum (FWHM) rather than the peak at its baseline for the calculations. The reason for that was to minimize the inﬂuence of slightly differently integrated peaks (thus, a potentially differently selected baseline) for the selection of the best-suited stationary phase. Finally, the chromatographic separation was performed on a Waters Acquity UPLC HSS T3 C18, 1.8 µm, 2.1 × 100 mm column at 25 ◦C with a ﬂow rate of 0.4 mL/min and 3 µL injection volume. Eluent A was composed of 5% ACN and eluent B of 98% ACN, both containing 2 mM ammonium acetate. The chromatographic separation of the analytes was achieved in a total run time of 7 min, with a gradient comprising an initial hold time of 0.5 min at 10% B and a linear gradient to 44% B within 3 min. The gradient was set to 100% B afterwards to wash the column till 5.4 min, followed to re-equilibration at 10% B until the end of the run. A switching valve directed the LC ﬂow to the MS from 1.0 to 3.8 min. Toxins 2021, 13, 600 10 of 12 10 of 12 Informed Consent Statement: Not applicable. Acknowledgments: We are grateful to DI Georg Häubl, Lilian Kuster and Ing. Günther Jaunecker (all Romer Labs, Tulln, Austria) for their support in the production and puriﬁcation of 13C-D3G. Open Access Funding by the Austrian Science Fund (FWF). Acknowledgments: We are grateful to DI Georg Häubl, Lilian Kuster and Ing. Günther Jaunecker (all Romer Labs, Tulln, Austria) for their support in the production and puriﬁcation of 13C-D3G. Open Access Funding by the Austrian Science Fund (FWF). Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. Conﬂicts of Interest: The authors declare no conﬂict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. 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The analytes concentrations were calculated by the relative response and the calibration curves with internal calibration. Apparent recoveries were calculated by the ratio of measured to spiked concentrations, followed by calculating the average value of all six spiking levels in triplicate analysis, expressed in percent. p p For the evaluation of matrix effects, the data were ﬁrst analyzed without considering the internal standards, which led to the determination of the apparent recoveries for external calibration. Furthermore, signal suppression or enhancement (SSE) of the SIDA method was calculated from the spiked blank extracts in the same way. To calculate the extraction recovery (RE), mean values of the apparent recovery using internal calibration (RA) were divided by the mean values of the signal suppression or enhancement (SSE). The repeatability (RSDr) was calculated from the triplicate analysis at seven spiking levels. Author Contributions: Conceptualization, G.A. and F.B.; methodology, I.F., H.M. and F.B.; validation, I.F. and F.B.; data curation, I.F. and M.M.S.; writing—original draft preparation, M.M.S. and F.B.; writing—review and editing, H.M. and G.A.; supervision, G.A. and F.B.; project administration and funding acquisition, G.A. and F.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Vienna Science and Technology Fund (WWTF LS12-021), the Austrian Science Fund (FWF; SFB F3706, F3708, and F3715) and Romer Labs (Tulln, Austria). Furthermore, ﬁnancial support by the Austrian Federal Ministry of Science, Research and Economy, Toxins 2021, 13, 600 11 of 12 11 of 12 the National Foundation for Research, Technology and Development, and BIOMIN Holding GmbH is acknowledged for funding the Christian Doppler Laboratory for Mycotoxin Metabolism. Institutional Review Board Statement: Not applicable. References 1. 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https://openalex.org/W4214546517	https://europepmc.org/articles/pmc6889710?pdf=render	English	null	Erratum: Docosahexaenoic acid is both a product of and a precursor to tetracosahexaenoic acid in the rat	Journal of lipid research	2,019	cc-by	147	Erratum: Docosahexaenoic acid is both a product of and a precursor to tetracosahexaenoic acid in the rat Erratum: Docosahexaenoic acid is both a product of and a precursor to tetracosahexaenoic acid in the rat Adam H. Metherel, R. J. Scott Lacombe, Raphaël Chouinard-Watkins, and Richard P. Bazinet Adam H. Metherel, R. J. Scott Lacombe, Raphaël Chouinard-Watkins, and Richard P. Bazinet Copyright © 2019 by the American Society for Biochemistry and Molecular Biology, Inc. Erratum Erratum Erratum VOL 60 (2019) PAGES 412–420 There was an error in the colors of the labels for Fig. 3A and B. The THA labels should be black and the DHA labels should be gray. This error does not affect the results or conclusions of this work. DOI https://doi.org/10.1194/jlr.ERR119000440 2102 Journal of Lipid Research Volume 60, 2019 2102 Journal of Lipid Research Volume 60, 2019 2102 Journal of Lipid Research Volume 60, 2019
https://openalex.org/W2776390275	http://ejournal.unp.ac.id/index.php/jippsd/article/download/8616/6615	Indonesian	null	PENGARUH STRATEGI GENIUS LEARNING DAN DISPOSISI MATEMATIS TERHADAP PEMAHAMAN KONSEP KELAS V SEKOLAH DASAR	Jurnal Inovasi Pendidikan Dan Pembelajaran Sekolah Dasar/Jurnal inovasi pendidikan dan pembelajaran sekolah dasar	2,017	cc-by	5,419	Abstrak Penelitian bertujuan untuk mengetahui: (1) pengaruhpemahaman konsep dengan strategi Genius Learning dibandingkan konvensional; (2) pengaruh pemahaman konsep dandisposisi matematis tinggi dengan strategi Genius Learning dibandingkan konvensional; (3) pengaruh pemahaman konsep dandisposisi matematis rendah dengan strategi Genius Learningdibandingkan konvensional; (4) interaksi antara strategi pembelajaran dengan disposisi matematis dalam mempengaruhi pemahaman konsep.Jenis penelitian yang digunakan adalah quasi eksperimen dengan rancangan penelitian menggunakan faktorial 2 x 2. Data penelitian diperoleh dari angket disposisi matematis dan tespemahaman konsep. Data dianalisis dengan uji-t dan Anava. Hasil penelitian menemukan bahwa: (1) pemahaman konsep dengan strategi Genius Learning lebih tinggi dibandingkankonvensional; (2) pemahaman konsep dandisposisi matematistinggi dengan strategi Genius Learninglebih tinggi dibandingkankonvensional; (3) pemahaman konsep dandisposisi matematisrendah denganstrategi Genius Learning lebih tinggi dibandingkankonvensional; (4) Tidak terdapat interaksi antara strategi pembelajaran dengan disposisi matematis dalam mempengaruhi pemahaman konsep. Kata Kunci: Pemahaman Konsep; Disposisi Matematis; Genius Learning Kata Kunci: Pemahaman Konsep; Disposisi Matematis; Genius Learning JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd PENGARUH STRATEGI GENIUS LEARNING DAN DISPOSISI MATEMATIS TERHADAP PEMAHAMAN KONSEP KELAS V SEKOLAH DASAR Feniareny. DA PGSD FKIP Universitas PGRI Palembang, Kota Palembang, Indonesia Email: feniareny@gmail.com Abstract This study was aimed at determining: (1) the effect of Genius Learning strategy toward concept understanding compared with conventional; (2) the effect of Genius Learning strategy towards concept understanding with high mathematical disposition compared with conventional; (3) the effect of Genius Learning strategy towards concept understanding with low mathematical disposition compared with conventional; (4) the interaction between learning strategy and mathematical disposition in influencing concept understanding. This study was designed as quasi-experimental research by using a 2 x 2 factorial. The data were obtained by using mathematical disposition questionnairesand concept understanding test. Data were analyzed by using t-test and Anova.The result of the data showed that: (1) understanding of the concepts taught by Genius Learning strategy was higher than conventional; (2) understanding of the concepts with high mathematical disposition taught by Genius Learning was higher than conventional; (3) understanding of the concepts with lower mathematical disposition taught by Genius Learning was higher than conventional; (4) There was no interaction between learning strategies andthemathematical disposition in influencing students’ understanding of the concept. Keywords: Concept Understanding; Mathematical Disposition; Genius Learning Copyright © 2017, JIPPSD, e-ISSN 2579-3403 74 Copyright © 2017, JIPPSD, e-ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 online at: http://e-journal.unp.ac.id/index Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Copyright © 2017, JIPPSD, e-ISSN 2579-3403 PENDAHULUAN mencari solusi alternatif untuk mengeksplorasi pengetahuan, ketekunan dalam memecahkan masalah matematika dan kemauan untuk merefleksikan pemikiran mereka sendiri ketika mereka belajar matematika. Indikator disposisi matematis dalam penelitian ini yaitu: (1) Rasa percaya diri; (2) Keingintahuan dan daya temu dalam melakukan tugas matematika; (3) Tekun mengerjakan tugas matematika; (4) Fleksibilitas dalam menyelidiki gagasan matematika dan berusaha mencari metode alternatif dalam memecahkan masalah; (5) Cenderung memonitor, merefleksikan performance penalaran sendiri. Matematika merupakan ilmu yang terdiri dari konsep-konsep, antara konsep yang satu dengan yang lainnya saling berkaitan atau berhubungan. Setiap orang mengenal matematika sebagai bidang studi yang memerlukan tingkat keterampilan lebih untuk bisa memahami konsep ilmu dan perhitungan dalam kegiatan pembelajarannya. Pemahaman konsep akan memudahkan siswa dalam mamahami materi dengan baik jika dalam menemukan konsep siswa diikutsertakan terlibat sendiri sehingga dengan begitu siswa akan lebih lama untuk mengingat dan lebih paham karena mengetahui bagaimana prosesnya. Indikator yang pemahaman konsep menunjukkan menurut Fadjar (2009:13) antara lain : (a) menyatakan ulang sebuah konsep; (b) mengklasifikasi objek menurut tertentu sesuai dengan konsepnya; (c) memberikan contoh dan bukan contoh dari suatu konsep; (d) menyajikan konsep dalam berbagai bentuk representasi matematis; (e) mengembangkan syarat perlu atau syarat cukup dari suatu konsep; (f) menggunakan dan memanfaatkan serta memilih prosedur atau operasi tertentu; (g) mengaplikasikan konsep atau algoritma dalam pemecahan masalah. Berdasarkan hasil observasi pada salah satu SD Gugus 2 Kecamatan Nanggalo, yaitu SDN 009 Surau Gadang, menunjukkan bahwa tujuan pembelajaran belum tercapai secara maksimal. Hal ini dilihat dari jawaban siswa pada ulangan harian. Salah satu contoh soal pemahaman konsep siswa dalam menggunakan dan memilih prosedur atau operasi tertentu adalah sebagai berikut: Gambar 1. Contoh soal pemahaman konsep Siswa Pemahaman konsep dipengaruhi oleh disposisimatematis yang dimiliki siswa. Menurut NCTM (2002) bahwa disposisi matematis merupakan kecenderungan untuk berpikir dan bertindak dengan cara yang positif seperti ketertarikan siswa dan kepercayaan diri dalam mengerjakan matematika, kemauan Gambar 1. Contoh soal pemahaman konsep Siswa Gambar 1. PENDAHULUAN menunjukkan bahwa siswa tidak dapat memilih prosedur yang benar dalam menyelesaikan konsep bangun ruang, artinya siswa belum paham tentang konsep Copyright © 2017, JIPPSD, e-ISSN 2579-3403 75 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd siswa akan lebih rileks dan tidak dibayang- bayangi dengan ketakutan dan kesulitan dalam mempelajari matematika itu sendiri sehingga siswa lebih berminat untuk belajar. Strategi genius learning akan membuat siswa merasa lebih lebih nyaman dalam memahami konsep matematika. Penerapan genius learning berangkat dari satu keyakinan dan pengharapan bahwa apabila setiap siswa dimotivasi dengan tepat dan diajar dengan cara yang benar, cara yang menghargai keunikan siswa maka siswa akan dapat mencapai pembelajaran yang maksimal (Khairani dan Filia, 2014: 161).Genius Learning memperdayakan siswa melalui 8 lingkaran sukses yaitu: suasana kondusif; hubungkan; big picture; goal setting; pemasukan informasi; aktivasi; demonstrasi; pengulangan dan jangkarkan. matematika dalam menyelesaikan soal yang diberikan. Berdasarkan observasi dan wawancara dengan guru di SDN 008 Surau Gadang, diperoleh sejumlah permasalahan yang disimpulkan: (1) kurangnya pemahaman siswa terhadap konsep dasar geometri, (2) pembelajaran geometri dipelajari siswa hanya di sekolah saja, sedangkan pada saat di rumah siswa jarang menerapkan pembelajaran geometri atau jarang berlatih mengerjakan soal-soal yang berhubungan dengan geometri, (3) siswa sering menghafal suatu konsep tanpa didasari dengan pemahaman, tidak mengetahui bagaimana proses mendapatkan rumus matematika sehingga siswa cepat lupa terhadap pembelajaran geometri yang sudah dijelaskan guru, (4) guru dalam menjelaskan materi geometri masih didomisili dengan menggunakan metode ceramah, dan (5) alat peraga yang dimiliki sekolah kurang lengkap. Menurut Gunawan (2007:3), bahwa strategi Genius Learning hasil yang dapat diperoleh untuk siswa yaitu dapat meningkatkan hasil belajar yang sangat signifikan (minimal 20%), suasana belajar yang lebih kondusif, siswa merasa lebih dihargai karena proses pembelajaran mengakomodasi semua gaya belajar mereka, motivasi belajar siswa meningkat, belajar menjadi suatu kegiatan yang menyenangkan, dan siswa mampu menerapkan apa yang mereka pelajari ke dalam kehidupan mereka (Gunawan: 2007:14-15). Untuk mengatasi permasalahan yang terjadi dalam pembelajaran matematika, hendaknya guru dapat menerapkan strategi pembelajaran yang tepat yang berorientasi kepada pemahaman siswa sehingga belajar menjadi aktif dan dinamis. Salah satu stategi pembelajaran yang melibatkan peran siswa secara aktif adalah Genius Learning. Copyright © 2017, JIPPSD, e-ISSN 2579-3403 Populasi dan Sampel Populasi dari penelitian ini adalah siswa kelas V SDN Kecamatan Nanggaloyang berjumlah 338 siswa. Pengambilan sampel dilakukan untuk pemilihan kelas eksperimen dan kelas kontrol pada penelitian ini secara Simple Random sampling. Dalam penelitian ini digunakan siswa kelas V SD Negeri 09 Surau Gadang sebagai kelas eksperimen dan siswa kelas V SD Negeri 08 sebagai kelas kontrol. METODE PENELITIAN Tahap persiapan dilaksanaan dengan langkah-langkah: (a) menentukan jadwal penelitian; (b) menentukan populasi dan sampel; (c) mempersiapkan Rencana pelaksanaan pembelajaran sesuai dengan strategi Genius Learning pada materi Geometri; (d) mempersiapkan Lembar Kerja Siswa; (e) mempersiapkan instrumen pengumpulan data; (f) Mempersiapkan angket; (g) memvalidasi semua perangkat penelitian; (h) melakukan uji coba semua perangkat penelitian; (i) melaksanakan analisis soal uji coba. Jenis Penelitian Jenis penelitian ini tergolong penelitian eksperimen semu (Quasi Experimental Design). Rancangan penelitian yang digunakan dalam penelitian ini untuk melihat pengaruh penerapan strategi Genius Learning terhadap pemahaman konsep adalah Posttest-Only Control Designmenurut Sugiyono (2012:112). Desain yang digunakan dalam penelitian ini adalah desain faktorial (2x2), dengan disposisi matematis sebagai variabel moderator. Desain penelitian ini seperti digambarkan dalam tabel 1 berikut. Data, Instrumen, dan Teknik Pengumpulan data Untuk memperoleh data yang diperlukan, penelitian ini menggunakan dua 2. Tahap pelaksanaan Tabel 1.Desain Penelitian Faktorial 2x2 Tabel 1.Desain Penelitian Faktorial 2x2 Tabel 1.Desain Penelitian Faktorial 2x2 Pemahaman Konsep Disposisi Matematis Genius Learning (A1) Konvensional (A2) Tinggi (B1) A1B1 A2B1 Rendah (B2) A1B2 A2B2 Sumber: Suryabrata (2006:119) Tahap pelaksanaan diawali dengan mengisi angket disposisi matematis kemudian melaksanakan pembelajaran di kelas eksperimen dengan strategi genius learningsedangkan untuk kelas kontrol menggunakan pembelajaran konvensional. 3. Tahap penutup Tahap penutup dilaksanaan dengan langkah-langkah: (a) Memberikan post-test pada kelas eksperimen dan kelas control; (b) Mengolah dan menganalisis data yang diperoleh dari kelas eksperimen dan kelas kontrol; (c) Menarik kesimpulan dan hasil yang diperoleh sesuai dengan analisis yang digunakan. PENDAHULUAN Strategi genius learning dalam pembelajaran membantu siswa untuk bisa mengerti kekuatan dan kelebihan mereka yang sesuai dengan gaya belajar mereka masing-masing. Dengan menerapkan strategi genius learning, proses belajar matematika Copyright © 2017, JIPPSD, e-ISSN 2579-3403 76 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Uji Persyaratan Analisis jenis instrumen yaitu instrumen tes dan instrument non-tes.. 1. Uji Normalitas. Uji normalitas yang digunakan adalah uji Lilliefors menurut Sudjana (2002: 466) 1. Tes, langkah-langkah penyusunan instrumen tes sebagai berikut: (a) membuat kisi-kisi soal; (b) menyusun butir-butir soal; (c) menyusun rublik penskoran tes; (d) memvalidasi soal tes melalui validator dengan memakai lembar validasi; dan (e) melakukan revisi. 2. Uji Homogenitas Variansi. Uji homogenitias variansi, dengan menggunakan rumus uji F dengan menggunakan rumus: s s F 2 2 2 1  2. Angket, langkah-langkah pembuatan angket disposisi matematis sebagai berikut: (a) menyusun kisi-kisi angket disposisi matematis; (b) menyusun item-item pernyataan angket disposisi matematis; (c) menvalidasi angket; (d) melakukan revisi angket setelah divalidasi oleh validator; dan (e) melakukan uji coba angket. Pengujian Hipotesis Untuk menguji hipotesis 1, 2, dan 3 digunakan uji-t karena membandingkan dua kelompok sampel, seperti yang dikemukakan leh Sudjana (2002:239) digunakan adalah dengan rumus : Teknik pengumpulan data untuk mengukur disposisi matematis siswa terhadap pembelajaran diberikan berupa angket. Fungsi pengambilan untuk menentukan variabel disposisi matematis, sehingga diperoleh kelompok siswa dengan disposisi matematis tinggi dan kelompok siswa dengan disposisi matematis rendah. Mengukur pemahaman konsep siswa terhadap materi luas trapesium dan layang-layang diberikan posttest. Soal pottest diberikan setelah siswa melakukan kegiatan pembelajaran dengan strategi pembelajaran genius learning dan pembelajaran konvensional. 2 1 2 1 1 1 n n S x x t    2 1 2 1 1 1 n n S x x t    2 1 2 1 1 1 n n S x x t    Dengan simpangan baku gabungan dicari dengan rumus:     2 1 1 2 1 2 2 2 2 1 1 2       n n s n s n S Kriteria pengujian adalah: apabila thitung> ttabel, maka Ho ditolak, dan H1 diterima, dengan 2 2 1    n n dk Kriteria pengujian adalah: apabila thitung> ttabel, maka Ho ditolak, dan H1 diterima, dengan 2 2 1    n n dk Uji hipotesis keempat menggunakan uji variansi (ANAVA) dua arah, dengan metode unweighted means. Prosedur Penelitian Prosedur dari penelitian ini adalah: Prosedur dari penelitian ini adalah: 1. Tahap persiapan Copyright © 2017, JIPPSD, e-ISSN 2579-3403 77 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Keterangan: g 1. Menyatakan ulang sebuah konsep; 2. Menggunakan dan memilih prosedur atau operasi tertentu; 2. Menggunakan dan memilih pros 3. Mengaplikasi konsep pemecahan masalah memperoleh rata-rata 3 dan kelas kontrol memperoleh rata-rata 1,8. Teknik Analisis Data Teknis analisis data dengan langkah- langkah sebagai berikut: Copyright © 2017, JIPPSD, e-ISSN 2579-3403 78 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd HASIL DAN PEMBAHASAN sedangkan rata-rata kontrol adalah 2,5. Perbedaan skor antara kelas ekperimen dan kelas kontrol sebanyak 0,8 untuk indikator pemahaman konsep nomor 2. Sedangkan untuk indikator soal ketiga kelas eksperimen memperoleh rata-rata 3,5 dan kelas kontrol memperoleh rata-rata 3. Ini dapat dilihat pada gambar 2. Perbedaan Pemahaman Konsep yang diajarkan dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Hasil tes pemahaman konsep siswa kelas eksperimen lebih tinggi dibandingkan siswa kelas kontrol jika dilihat masing-masing indikator soal pemahaman konsep. Indikator pertama rata-rata skor kelas eksperimen 3,2 Gambar 2. Diagram Batang Rata-rata Indikator Pemehaman Konsep 0 1 2 3 4 1 2 3 Rata-rata Skor Eksperimen Kontrol Gambar 2. Diagram Batang Rata-rata Indikator Pemehaman Konsep Keterangan: Uji Normalitas Pemahaman konsep siswa kelas eksperimen memiliki Lhitung= 0,1097 dan kelas kontrol memiliki Lhitung = 0,1179 pada taraf nyata 0,05. Pemahaman konsep siswa pada disposisi matematis tinggi kelas eksperimen memiliki Lhitung= 0,1515dan kelas kontrol memiliki Lhitung = 0,0985 dan pemahaman konsep siswa pada disposisi matematis rendah kelas eksperimen memiliki Lhitung= 0,1609 dan kelas kontrol memiliki Lhitung = 0,1863 pada taraf nyata 0,05. Dapat disimpulkan bahwa data pemahaman konsep siswa kelas eksperimen dan kelas kontrol baik secara keseluruhan, kelompok siswa yang mempunyai disposisi matematis tinggi dan disposisi matematis rendah kelas eksperimen maupun kelas kontrol berdistribusi normal, sebab Lhitung lebih kecil dari Ltabel. JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd pemahaman konsep nomor 2. Sedangkan untuk indikator soal ketiga kelas eksperimen memperoleh rata-rata 2 dan kelas kontrol memperoleh rata-rata 1,8. dan pemahaman konsep siswa pada disposisi matematis rendah kelas eksperimen dan kelas kontrol memiliki Fhitung = 0,66 dan Ftabel = 2,03 pada taraf nyata 0,05. Dapat disimpulkan bahwa data pemahaman konsep siswa kelas eksperimen dan kelas kontrol baik secara keseluruhan, kelompok siswa yang mempunyai disposisi matematis tinggi dan disposisi matematisrendah kelas eksperimen maupun kelas kontrol bervarians homogen, sebab Fhitung< Ftabel. Copyright © 2017, JIPPSD, e-ISSN 2579-3403 Perbedaan Pemahaman Konsep dengan Disposisi Matematis Tinggi yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Perbedaan Pemahaman Konsep dengan Disposisi Matematis Tinggi yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Perbedaan Pemahaman Konsep dengan Disposisi Matematis Tinggi yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Perbedaan Pemahaman Konsep dengan Disposisi Matematis Rendah yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Rara-rata skor indiktor pemahaman konsep siswa berkemampuan disposisi matematis tinggi pada kelas eksperimen dan kelas kontrol jika dilihat masing-masing indikator soal pemahaman konsep. Indikator pertama rata-rata skor kelas eksperimen 3,3 sedangkan rata-rata kontrol adalah 2,5. Perbedaan skor antara kelas ekperimen dan kelas kontrol sebanyak 0,7 untuk indikator pemahaman konsep nomor 2. Sedangkan untuk indikator soal ketiga kelas eksperimen Rara-rata skor indiktor pemahaman konsep siswa berkemampuan disposisi matematis rendah pada kelas eksperimen dan kelas kontrol jika dilihat masing-masing indikator soal pemahaman konsep. Indikator pertama rata-rata skor kelas eksperimen 2,2 sedangkan rata-rata kontrol adalah 2. Perbedaan skor antara kelas ekperimen dan kelas kontrol sebanyak 0,2 untuk indikator Copyright © 2017, JIPPSD, e-ISSN 2579-3403 79 1. Pemahaman Konsep Siswa Kelas Eksperimen dan Kelas Kontrol Hasil perhitungan dengan menggunakan Uji-t diperoleh thitung = 10,69 pada taraf α= 0,05 diperoleh ttabel = 1,660 karena thitung > ttabel H0 ditolak. Ini berarti pemahaman konsep geomertri yang diajar dengan Genius learning lebih tinggi dari pada pemahaman konsep siswa yang diajar dengan konvensional. 2. Pemahaman Konsep Siswa dengan Disposisi Matematis Tinggi Kelas Eksperimen dan Kelas Kontrol 4. Interaksi antara Strategi Pembelajaran dengan Disposisi matematis dalam mempengaruhi Pemahaman Konsep. 4. Interaksi antara Strategi Pembelajaran dengan Disposisi matematis dalam mempengaruhi Pemahaman Konsep. g Untuk menarik perhatian siswa guru menghubungkan antara materi yang akan dipelajari dengan pengetahuan yang telah dimiliki siswa, baik itu dari proses pembelajaran sebelumnya maupun dari pengalaman siswa itu sendiri. Saat guru berhasil menghubungkan antara materi yang akan dipelajari dengan apa yang telah diketahui oleh murid, maka akan terjadi kesiapan dalam diri murid. Cara yang paling mudah dapat dilakukan adalah dengan mengajukan pertanyaan. Hipotesis keempat menggunakan Uji ANAVA dua arah dengan metode Unweighted mean. Bahwa nilai Fhitung = 0,28 < Ftabel = 3,938. Hal ini berarti H0 diterima, dan disimpulkan bahwa sesungguhnya tidak terdapat interaksi antara strategi pembelajaran dan disposisi matematis terhadap pemahaman konsep siswa. 3. Gambaran besar (Big Picture) Untuk membantu menyiapkan pikiran murid dalam menyerap materi yang akan diajarkan, sebelum proses pembelajaran dimulai guru harus memberikan gambaran besar dari keseluruhan materi. Memberikan gambaran besar ini berfungsi sebagai perintah kepada pikiran untuk menciptakan “folder” yang nantinya akan di isi dengan informasi. Gambaran besar (Big Picture) Untuk membantu menyiapkan pikiran murid dalam menyerap materi yang akan diajarkan, sebelum proses pembelajaran dimulai guru harus memberikan gambaran besar dari keseluruhan materi. Perbedaan Pemahaman Konsep dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional 3. Pemahaman Konsep Siswa dengan Disposisi Matematis Rendah Kelas Eksperimen dan Kelas Kontrol 3. Pemahaman Konsep Siswa dengan Disposisi Matematis Rendah Kelas Eksperimen dan Kelas Kontrol Hasil perhitungan dengan menggunakan Uji-t diperoleh thitung = 10,34 pada taraf nyata α = 0,05 diperoleh ttabel = 1,677 karena thitung> ttabel, H0 ditolak. Ini berarti pemahaman konsep dan disposisi matematis rendah yang diajar dengan strategi Genius learning lebih tinggi dari pada pemahaman konsep siswa yang diajar dengan konvensional. 2. Hubungkan Hubungkan Untuk menarik perhatian siswa guru menghubungkan antara materi yang akan dipelajari dengan pengetahuan yang telah dimiliki siswa, baik itu dari proses pembelajaran sebelumnya maupun dari pengalaman siswa itu sendiri. Saat guru berhasil menghubungkan antara materi yang akan dipelajari dengan apa yang telah diketahui oleh murid, maka akan terjadi kesiapan dalam diri murid. Cara yang paling mudah dapat dilakukan adalah dengan mengajukan pertanyaan. Copyright © 2017, JIPPSD, e-ISSN 2579-3403 Uji Homogenitas Hasil perhitungan dengan menggunakan Uji-t diperoleh thitung = 7,32 pada taraf nyata α = 0,05 di peroleh ttabel = 1,676 karena thitung> ttabel, H0 ditolak. Ini berarti pemahaman konsepdan disposisi matematis tinggi yang di ajar dengan strategi Genius learning lebih tinggi dari Hasil perhitungan dengan uji F bahwa pemahaman konsep siswa kelas eksperimen dan kelas kontrol memiliki Fhitung= 0,48 dan Ftabel = 1,604 pada taraf nyata 0,05. Pemahaman konsep siswa pada disposisi matematis tinggi kelas eksperimen dan kelas kontrol memiliki Fhitung= 0,38 dan Ftabel = 1,94 Copyright © 2017, JIPPSD, e-ISSN 2579-3403 80 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd pada pemahaman konsep siswa yang diajar dengan konvensional. pada pemahaman konsep siswa yang diajar dengan konvensional. Suasana kondusif Suasana yang kondusif merupakan suasana yang mendukung proses pembelajaran. Lingkungan yang mendukung sangat menentukan keberhasilan suatu proses pembelajaran, karena tanpa tanpa hal itu strategi apa pun yang diterapkan di dalam kelas akan sia-sia. Guru bertanggungjawab untuk menciptakan iklim belajar yang kondusif sebagai persiapan untuk masuk ke dalam proses pembelajaran yang sebenarnya. Selain itu guru perlu menunjukkan pengharapan yang besar terhadap keberhasilan siswa dan memastikan siswa tidak takut untuk membuat kesalahan dengan memberikan pengertian bahwa kesalahan merupakan bagian dari proses pembelajaran. Copyright © 2017, JIPPSD, e-ISSN 2579-3403 Perbedaan Pemahaman Konsep dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Tetapkan tujuan Pada tahap ini, hasil yang akan dicapai pada akhir sesi harus dijelaskan dan dinyatakan kepada siswa. Dengan mengetahui tujuan dari proses pembelajaran yang akan dilaksanakan, Penelitian ini menggunakan strategi pembelajaran Genius.Menurut Gunawan, Adi W (2007: 334-357) menjelaskan ada delapan langkah penerapan GeniusLearning sebagai berikut: Copyright © 2017, JIPPSD, e-ISSN 2579-3403 81 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd siswa akan termotivasi untuk aktif dalam pembelajaran. jumlah siswa bisa menyajikan konsep dalam berbagai bentuk representasi matematis, hanya ada sekitar 2 orang yang masih belum menjawab dengan benar, dan ada 9 orang hampir menjawab dengan benar. siswa akan termotivasi untuk aktif dalam pembelajaran. 5. Pemasukan informasi Pada tahap ini, guru menyampaikan materi pembelajaran dengan menggunakan strategi yang cocok dengan karakteristik dan gaya belajar siswa. Indikator pemahaman konsep selanjutnya adalah menggunakan dan memilih prosedur atau operasi tertentu yang ada pada soal nomor 2 yaitu “Hitung luas trapesium berikut. Jika a = 80 m, b = 100 m dan t = 61 m?”. Dari jawaban siswa dapat dilihat bahwa hanya ada 6 orang siswa yang tidak bisa menjawab, hal ini mungkin dikarenakan pemanfaatan waktu siswa yang kurang atau siswa masih ragu dengan langkah penyelesainnya. Jawaban lain ada 2 orang siswa yang menjawab dengan separuh benar dan selebihnya sudah hampir benar dalam menjawb soal. 6. Aktivasi 6. Aktivasi Proses aktivasi merupakan proses yang membawa murid kepada satu tingkat pemahaman yang lebih dalam terhadap materi yang diajarkan. Aktivasi dapat dilakukan secara perorangan, berpasangan, ataupun secara berkelompok. Dengan cara memberikan tugas ataupun memberikan masalah- masalah yang harus dipecah kan seputar materi pembelajaran. 7. Demonstrasi Tahap ini sebenarnya sama dengan proses guru menguji pemahaman murid dengan memberikan ujian. Bedanya dalam lingkaran sukses Genius Learning, kita lansung menguji pemahaman murid saat itu juga, ini bertujuan untuk benar-benar mengetahui sampai dimana pemahaman murid dan sekaligus merupakan saat yang tepat untuk memberikan umpan balik. Demonstrasi dapat dilakukan dengan cara siswa mempresentasikan jawaban dari tugas dan masalah yang telah mereka pecahkan. Indikator pemahaman konsep ketiga yang dilihat dalam penelitian ini adalah mengaplikasi konsep atau alogaritma pemecahan masalah pada soal nomor 7 yaitu “Selembar kertas berbentuk trapesium sama sisi dengan ukuran sisi yang sejajar 24 dm dan 16 dm. Luas trapesium adalah 400 dm2. Berapa Tinggi trapesium tersebut?” Hasil dari jawaban siswa pada indikator ini adalah 2 orang siswa menjawab dengan asal, 6 orang siswa menjawab mulai hampir sesuai dengan maksud soal sedangkan selebihnya siswa mulai menjawab benar dan hampir mendekati benar. 8. Ulangi (review) dan jangkarkan Lakukan pengulangan dan penjangkaran pada akhir setiap sesi dan sekaligus membuat kesimpulan dari apa yang telah dipelajari. Ini bermanfaat untuk meningkatkan daya ingat dan meningkatkan efektifitas dari proses pembelajaran. Berdasarkan tes akhir siswa kelas eksperimen hampir semua mampu menguasai indikator pemahaman konsep misalkan pada soal 1 yaitu “Bagaimana mencari rumus luas trapesium dengan mengggunakan rumus persegi panjang?”. Lebih dari separuh dari Kesalahan siswa dalam memahami soal adalah siswa kurang memahami maksud dari “sisi yang sejajar 24 dm dan 16 dm” yang ada pada Copyright © 2017, JIPPSD, e-ISSN 2579-3403 82 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Volume 1, Nomor 2, Desember 2017 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd , , Available online at: http://e-journal.unp.ac.id/index.php/jippsd soal, siswa belum bisa menggambarkan maksud dari soal tersebut. Sedangkan pada kelas kontrol hanya ada 3 orang siswa yang mulai menjawab benar meskipun secara keseluruhan belum benar. Copyright © 2017, JIPPSD, e-ISSN 2579-3403 6. Aktivasi lingkaran sukses genius learning yaitu goal setting (penetapan tujuan) dianggap berperan dalam membantu siswa mengembangkan indikator disposisi matematis tersebut dalam diri siswa. Siswa diminta untuk memahami citra dirinya atau mengetahui sejauh mana kemampuaanya, kemudian melalui tahap goal pada goal setting siswa merancang target yang ingin diraihnya atau dengan kata lain menciptakan diri ideal, langkah selanjutnya siswa diminta merancang, melaksanakan dan meninjau tugas yang telah disusun setiap hari untuk mewujudkan target atau goal. Semakin konsisten melaksanakan tugas dalam mewujudkan goal, maka akan semakin tinggi disposisi matematis yang positif dan merupakan unsur penting untuk memcapai keberhasilan (Gunawan, 2012) Perbedaan Pemahaman Konsep dengan Disposisi Matematis Tinggi yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Pembelajaran strategi Genius learning mampu meningkatkan disposisi matematis dan minat belajar siswa. Hal ini terjadi karena strategi genius leaning adalah suatu rangkaian pendekatan paraktis dalam mengingkatkan proses pembelajaran yang dicapai dengan menggunakan pengetahuan yang berasal dari berbagai disiplin ilmu salah satunya adalah disposisi matematis (Gunawan, 2012). Strategi genius learning membantu siswa mengubah dan mengikatkan disposisi matematis dengan mengintergrasikan teknik goal setting (penetapan tujuan) langsung pada lingkaran suksesnya seperti yang dikemukakan Gunawan (2012). Dalam goal setting tersebut siswa kan menentukan sendiri goal atau target yang ingin mereka capai dan membuat perencanaan tugas yang akan mereka lakukan guna mencapai goal. rencana tersebut harus mereka lakukan dan tinjau setiap hari. Dalam Strategi genius learning melalui lingkaran sukses “pemasukan informasi”, digunakan Lembar Kerja Siswa (LKS). LKS dikerjakan oleh siswa dengan berdiskusi secara berkelompok beranggotakan 4 sampai 5 orang yang heterogen. LKS disusun berorientasi pada strategi genius leaning yang dirancang untuk mengembangkan gaya belajar pada diri siswa. Bagian awal dari LKS berisikan fakta-fakta menarik yang ditemui siswa dalam kehidupan sehari-hari yang berguna, sehingga dapat mengoptimalkan gaya belajar. Siswa diminta siswa diminta untuk memahami sejauh mana citra dirinya atau sejauh mana kemampuannya melalui teknik membaca. Selanjutnya siswa diminta merancang dan melaksanakan percobaan, berupa langkah-langkah kegiatan Dalam Strategi genius learning melalui lingkaran sukses “pemasukan informasi”, digunakan Lembar Kerja Siswa (LKS). LKS dikerjakan oleh siswa dengan berdiskusi secara berkelompok beranggotakan 4 sampai 5 orang yang heterogen. LKS disusun berorientasi pada strategi genius leaning yang dirancang untuk mengembangkan gaya belajar pada diri siswa. Perbedaan Pemahaman Konsep dengan Disposisi Matematis Rendah yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Grafik Interaksi antara Strategi Pembelajaran dan Dispisisi Matematis dalam Mempengaruhi Pemahaman Konsep Matematika Siswa 0 20 40 60 80 100 Disposisi Matematis Tinggi Disposisi Matematis Rendah Eksperimen Kontrol JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 yang menuntut siswa. Semakin konsisten melaksanakan percobaan dalam mewujudkan gaya belajar auditori maka akan semakin tinggi disposisi matematis yang positif dan merupakan unsur penting untuk memcapai keberhasilan. langkah selanjutnya adalah kesimpulan, melalui bagian ini, siswa mendengarkan dan memberi tanggapan untuk menyimpulkan konsep yang baru ditemukan dengan konsep yang telah mereka kenal sebelumnya. Interaksi antara dua variabel bebas bisa terjadi apabila terjadi pengaruh yang berbeda dari salah satu diantara kedua variabel bebas itu pada tingkatan yang berbeda dari variabel lainnya. Grafiknya membentuk dua buah garis yang saling sejajar. Hal ini sesuai dengan pendapat Ary (1982:365) yang mengatakan untuk menilai interaksi antara kedua variable bebas dapat ditunjukkan secara grafis, jika digrafik diperoleh kedua garis saling berpotongan maka terdapat interaksi, tetapi jika kedua garis saling sejajar maka tidak terdapat interaksi. Dapat disimpulkan bahwa tidak terdapat interaksi antara strategi pembelajaran dengan disposisi matematis dalam mempengaruhi pemhamaan konsep matematika. Untuk mereview hasil kerja siswa pada LKS, melalui strategi genius leaning, guru meminta siswa untuk “mendemonstasikan” temuannya. Siswa diminta mendengarkan dan memberi tanggapan hasil kerja yang telah ditemukan. Sehingga dapat meningkatkan disposisi matematis. Interaksi antara Strategi Pembelajaran dan Disposisi Matematis terhadap Pemahaman Konsep Berdasarkan data dengan menggunakan strategi pembelajaran Genius learning nilai rata-rata kemampuan pemahaman konsep matematika siswa disposisi matematis tinggi 87,49 dan rata-rata pemahaman konsep matematika siswa disposisi matematis rendah 75. Dengan menggunakan pembelajaran konvensional rata-rata pemahaman konsep masalah matematika siswa disposisi matematis tinggi 50,47 dan rata-rata pemahaman konsep matematika siswa disposisi matematis rendah 35. Secara grafik dapat digambarkan seperti berikut: Perbedaan Pemahaman Konsep dengan Disposisi Matematis Rendah yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Perbedaan Pemahaman Konsep dengan Disposisi Matematis Rendah yang diajar dengan Strategi Genius Learning dan Strategi Pembelajaran Konvensional Dalam penelitian ini, untuk membangun disposisi matematis yang lebih positif, diterapkan strategi genius learning. Salah satu Copyright © 2017, JIPPSD, e-ISSN 2579-3403 83 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd yang menuntut siswa. Semakin konsisten melaksanakan percobaan dalam mewujudkan gaya belajar auditori maka akan semakin tinggi disposisi matematis yang positif dan merupakan unsur penting untuk memcapai keberhasilan. langkah selanjutnya adalah kesimpulan, melalui bagian ini, siswa mendengarkan dan memberi tanggapan untuk menyimpulkan konsep yang baru ditemukan dengan konsep yang telah mereka kenal sebelumnya. Untuk mereview hasil kerja siswa pada LKS, melalui strategi genius leaning, guru meminta siswa untuk “mendemonstasikan” temuannya. Siswa diminta mendengarkan dan memberi tanggapan hasil kerja yang telah ditemukan. Sehingga dapat meningkatkan disposisi matematis. Interaksi antara Strategi Pembelajaran dan Disposisi Matematis terhadap Pemahaman Konsep Berdasarkan data dengan menggunakan Gambar 3. Grafik Interaksi antara Strategi Pembelajaran dan Dispisisi Matematis dalam Mempengaruhi Pemahaman Konsep Matematika Siswa Interaksi antara dua variabel bebas bisa terjadi apabila terjadi pengaruh yang berbeda dari salah satu diantara kedua variabel bebas itu pada tingkatan yang berbeda dari variabel lainnya. Grafiknya membentuk dua buah garis yang saling sejajar. Hal ini sesuai dengan pendapat Ary (1982:365) yang mengatakan untuk menilai interaksi antara kedua variable bebas dapat ditunjukkan secara grafis, jika digrafik diperoleh kedua garis saling berpotongan maka terdapat interaksi, tetapi jika kedua garis saling sejajar maka tidak 0 20 40 60 80 100 Disposisi Matematis Tinggi Disposisi Matematis Rendah Eksperimen Kontrol JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd yang menuntut siswa. Semakin konsisten melaksanakan percobaan dalam mewujudkan gaya belajar auditori maka akan semakin tinggi disposisi matematis yang positif dan merupakan unsur penting untuk memcapai keberhasilan. langkah selanjutnya adalah kesimpulan, melalui bagian ini, siswa mendengarkan dan memberi tanggapan untuk menyimpulkan konsep yang baru ditemukan dengan konsep yang telah mereka kenal Gambar 3. Grafik Interaksi antara Strategi Pembelajaran dan Dispisisi Matematis dalam Mempengaruhi Pemahaman Konsep Matematika Siswa 0 20 40 60 80 100 Disposisi Matematis Tinggi Disposisi Matematis Rendah Eksperimen Kontrol EMBELAJARAN SEKOLAH DASAR 403 Desember 2017 l.unp.ac.id/index.php/jippsd Gambar 3. SIMPULAN Berdasarkan hasil analisis uji-t pada taraf signifikan α = 0,05 diperolehthitung>ttabel(thitung= 10,69 >ttabel = 1,660), sehingga dapat disimpulkan ada pengaruh yang signifikan dari strategi genius Copyright © 2017, JIPPSD, e-ISSN 2579-3403 84 JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd JURNAL INOVASI PENDIDIKAN DAN PEMBELAJARAN SEKOLAH DASAR ISSN 2579-3403 Volume 1, Nomor 2, Desember 2017 , , Available online at: http://e-journal.unp.ac.id/index.php/jippsd Available online at: http://e-journal.unp.ac.id/index.php/jippsd DAFTAR RUJUKAN learningterhadap pemahaman konsep siswa kelas V Kecamatan Nanggalo. Arnidha, Y. (2013). Efektifitas Model Kooperatif Think Pair Share Terhadap Kemampuan Pemahaman Konsep dan Disposisi Matematis Siswa. Tesis Tidak Dipublikasikan. Jakarta: Universitas Terbuka. Berdasarkan hasil analisis uji-t pada taraf signifikan α = 0,05 diperolehthitung>ttabel(thitung= 7,32 >ttabel= 1,676), sehingga dapat disimpulkanada pengaruh yang signifikan dari strategi genius learningdan disposisi matematis tinggi terhadap pemahaman konsep siswa kelas V Kecamatan Nanggalo. Ary, D. (1982). Pengantar Penelitian dan Pendidikan, Surabaya: UsahaNasional Depdiknas. (2001). Penyusunan Butir-butir Soal dan Instrumen Penelitian. Jakarta: Depdiknas. Fadjar, S. (2009). Kemahiran Matematika. Yogyakarta: Depdiknas. Berdasarkan hasil analisis uji-t pada taraf signifikan α = 0,05 diperoleh thitung > ttabel (thitung =10,34 > ttabel = 1,677), sehingga dapat disimpulkan ada pengaruh yang signifikan dari strategi genius learningdan disposisi matematis rendah terhadap pemahaman konsepsiswa kelas V Kecamatan Nanggalo. Gunawan, A. W. (2012). Genius Learning Strategi Petunjuk Praktis untuk Menerapkan Accelereted Learning. Jakarta: Gremedia Pustaka Utama. Katz. L.G. (1993). Dispositions as Educational Goals. New York. Forguson. Kesumawati, N. (2010). Peningkatan Kemampuan Pemahaman, Pemecahan Masalah dan Disposisi Matematis Siswa SMP Melalui Pendekatan 79 Pendidikan Matematika Realistik. Disertasi tidak diterbitkan. Bandung: Doktor pada SPs UPI. Berdasarkan hasil analisis Anava dua arahdiperoleh tabel hitung F F  (Fhitung = 0,28 <Ftabel = 3,938,sehingga dapat disimpulkantidak terdapat interaksi antara strategi pembelajaran Genius learning dan disposisi matematis terhadap pemahaman konsep siswa kelas V Kecamatan Nanggalo. Khairani, M.L dan Sari, R.F. (2014). Strategi Genius Learning dalam Pembelajaran Matematika. Medan: Fakultas Ilmu Tarbiyah dan Keguruan IAIN Sumatera Utara. Maxwell, K. (2001). Positive Learning Dispositions in Mathematics. New York: Macmillan International. National Council of Teahers of Mathematic. (2002). Currikulum and Evaluation’s Standards’ for School Mathematics. Reston, Virginia: NCTM. Sanjaya, W. (2011). Strategi Pembelajaran Berorientasi Standar Proses Pendidikan. Jakarta: Kencana. Copyright © 2017, JIPPSD, e-ISSN 2579-3403 Copyright © 2017, JIPPSD, e-ISSN 2579-3403 85 Volume 1, Nomor 2, Desember 2017 Available online at: http://e-journal.unp.ac.id/index.php/jippsd Pengembangan Pengajaran. Jakarta: Erlangga Sugiyono. (2012). Metode Penelitian Pendidikan Pendekatan Kuantitatif, Kualitatif, dan R & D. Bandung: Alfabeta PROFIL SINGKAT Sudjana. (2002). Metode Statistika. Bandung: Tarsito. Feniareny.DA.,M.Pd. dilahirkan di kota Lubuklinggau (Sumsel) tanggal 04 Februari 1989. Pendidikan S1 ditempuh di Universitas Sriwijaya Prodi PGSD FKIP lulus tahun 2011 dan melanjutkan studi S2 di Universitas Negeri Padang lulus tahun 2017 pada prodi Pendidikan Dasar. Saat ini pekerjaan yang dijalankan menjadi seorang dosen Prodi PGSD FKIP Universitas PGRI Palembang. Sumarno, U. (2013). Berfikir dan Disposisi Matematika serta Pembelajarannya. Bandung : Univ. Pendidikan Indonesia. Suryabrata, S. (2006). Metode Penelitian. Jakarta: Raja Grafindo Prasada. Van De Walle, J. A. (2008). Matematika Sekolah Dasar dan Menengah Sumarno, U. (2013). Berfikir dan Disposisi Matematika serta Pembelajarannya. Bandung : Univ. Pendidikan Indonesia. Suryabrata, S. (2006). Metode Penelitian. Jakarta: Raja Grafindo Prasada. Van De Walle, J. A. (2008). Matematika Sekolah Dasar dan Menengah Copyright © 2017, JIPPSD, e-ISSN 2579-3403 Copyright © 2017, JIPPSD, e-ISSN 2579-3403 86
https://openalex.org/W96562470	https://tobaccoinduceddiseases.biomedcentral.com/counter/pdf/10.1186/1617-9625-12-S1-A11	English	null	Periodontal disease of secondhand smoking patients	Tobacco induced diseases	2,014	cc-by	815	Authors’ details 1 1Department of Periodontology, Faculty of Dentistry, Osmangazi University, Eskisehir, 26480, Turkey. 2Institute of Biomedical Engineering, Bogazici University, Istanbul, 34684, Turkey. 3Medico-social Centre, Dental Clinic, Bogazici University, Istanbul, 34684, Turkey. 4Tepebasi Oral and Dental Health Hospital, Ankara, 06400, Turkey. Results Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit This is an Open Access article distributed under the terms of the Creative Commons licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in l it d Th C ti C P bli D i D di ti i (htt // Published: 6 June 2014 doi:10.1186/1617-9625-12-S1-A11 Cite this article as: Beklen et al.: Periodontal disease of secondhand smoking patients. Tobacco Induced Diseases 2014 12(Suppl 1):A11. doi:10.1186/1617-9625-12-S1-A11 Cite this article as: Beklen et al.: Periodontal disease of secondhand smoking patients. Tobacco Induced Diseases 2014 12(Suppl 1):A11. Background The authors declare no conflict of interests and thank their institutes for the support. The skillful technical help of Dr. Elif B. Barto is thankfully acknowledged. Our cross-sectional study investigated associations of cumulative exposure to secondhand smoke and duration of smoking cessation with periodontitis among family members. Furthermore the effects of other risk factors on these associations were analyzed as well. Beklen et al. Tobacco Induced Diseases 2014, 12(Suppl 1):A11 http://www.tobaccoinduceddiseases.com/content/12/S1/A11 MEETING ABSTRACT Open Access * Correspondence: arzubeklen@yahoo.com 1Department of Periodontology, Faculty of Dentistry, Osmangazi University, Eskisehir, 26480, Turkey Full list of author information is available at the end of the article Materials and methods In total 109 patients were analyzed with their full mouth periodontal examination. The groups were divided as cur- rent second hand smokers, former second hand smokers and non-smokers. The associations between periodontitis occurrence and potential risk factors was analyzed using univariate and multivariate analysis. Published: 6 June 2014 Results The results showed that the rates of periodontitis among nonsmokers, former second hand smokers, and current second hand smokers were 19.5%, 22.3%, 37.4% respec- tively. We then adjusted the other periodontal risk factors and we found that the odds ratio (95% confidence interval) for periodontitis was 2.12 for former second-hand smokers and 3.56 for current second hand smokers. Conclusions In summary, not only a significant dose-response relation- ship between pack-years of second hand smoking and per- iodontitis presence was observed, but also a significant decrease in the occurrence of periodontitis was observed in second hand smoke stopped group. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit * Correspondence: arzubeklen@yahoo.com 1Department of Periodontology, Faculty of Dentistry, Osmangazi University, Eskisehir, 26480, Turkey Full list of author information is available at the end of the article © 2014 Beklen 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/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit This is an Open Access article distributed under the terms of the Creative Commons /licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in perly cited. The Creative Commons Public Domain Dedication waiver (http:// pplies to the data made available in this article, unless otherwise stated. Conclusions In summary, not only a significant dose-response relation- ship between pack-years of second hand smoking and per- iodontitis presence was observed, but also a significant decrease in the occurrence of periodontitis was observed in second hand smoke stopped group. • Convenient online submission * Correspondence: arzubeklen@yahoo.com 1Department of Periodontology, Faculty of Dentistry, Osmangazi University, Eskisehir, 26480, Turkey Full list of author information is available at the end of the article © 2014 Beklen 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/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
W3006685213.txt	null	en		Personality associations with Facebook use and tendencies towards Facebook Use Disorder	Addictive behaviors reports	2,020	cc-by	8,970	Addictive Behaviors Reports 11 (2020) 100264 Contents lists available at ScienceDirect Addictive Behaviors Reports journal homepage: www.elsevier.com/locate/abrep Personality associations with Facebook use and tendencies towards Facebook Use Disorder T Cornelia Sindermanna,⁎, Éilish Dukeb, Christian Montaga a b Department of Molecular Psychology, Institute of Psychology and Education, Ulm University, 89081 Ulm, Germany Department of Psychology, University of Huddersfield, Queensgate, Huddersfield HD1 3DH, UK ARTICLE INFO ABSTRACT Keywords: Facebook Big Five Personality Facebook Use Disorder Internet Communication Disorder Social Networks Use Disorder Introduction: The present study sought to address core issues in the association between personality and the putative disordered use of Facebook. First, to redress the issue of generalisation from samples solely recruited from Facebook, we sought to explore personality differences between users and non-users of Facebook. Second, we aimed to investigate associations between personality and Facebook Use Disorder. The present study contributes a novel perspective to extant research on this topic by moving beyond the broad Big Five of personality, to explore possible relationships between Facebook use and sub-facets of the Big Five; all analyses were additionally controlled for confounding effects of demographic variables. Methods: 3,835 (n = 2,366 males) participants completed socio-demographic variables, the Big Five Inventory and stated their user status on Facebook (i.e. user versus non-user). Facebook-users also completed a Facebook Use Disorder scale assessing addictive tendencies towards Facebook use. Results: Facebook users reported higher levels of extraversion and lower levels of conscientiousness compared to non-users. Tendencies towards Facebook Use Disorder correlated negatively with conscientiousness and positively with neuroticism in both males and females. Conclusions: The present results indicate that research samples drawn from Facebook users may be biased with regard to personality (extraversion, conscientiousness). Moreover, certain personality traits – conscientiousness and neuroticism – which may influence the tendency towards Facebook Use Disorder are discussed. 1. Introduction 1.1. General introduction Use of online social networks (SNs) such as Facebook, Twitter, and Instagram is an increasingly common aspect of modern life. Facebook, which boasts approximately 2,375 million active users, is one of the largest SNs worldwide (We Are Social, Hootsuite, & DataReportal, 2019). Reflecting this popularity, the Facebook application is one of the most frequently downloaded social (networking) apps from Apple’s app store and Google’s Play store, suggesting that a large proportion of people have constant access to Facebook via their smartphone (Priori Data, 2019a,b). Given its popularity, Facebook affords researchers access to a large number of prospective participants. However, sole reliance on Facebook as a recruitment platform may lead to biases in sampling, particularly if robust individual differences, e.g. in personality traits, exist between users and non-users. Personality can be defined as “stable ⁎ individual differences in cognitive, emotional and motivational aspects of mental states that result in stable behavioral action (especially emotional) tendencies of humans …” (Montag & Panksepp, 2017, p. 1). Given this definition, it seems logical that certain personality traits may be associated with whether or not people use Facebook. Therefore, if only recruiting Facebook users, such differences between users and non-users may lead to the publication of data that cannot be generalised beyond the sample of Facebook users (Brickman Bhutta, 2012; Thornton et al., 2016; Whitaker, Stevelink, & Fear, 2017). It is, therefore, important to investigate potential systematic differences between Facebook users and non-users in personality, which was the first aim of the present study. Next, potential addictive tendencies towards the use of Internet activities such as the use of SNs like Facebook have been investigated in several studies (see for example studies mentioned in paragraph 2.2). Specifically, the putative addictive tendencies toward the Internet have been parsed into generalised and specific activities (Davis, 2001). According to Davis’ model, specific Internet Use Disorders do not refer to Corresponding author at: Department of Molecular Psychology, Ulm University, Helmholtzstraße 8/1, 89081 Ulm, Germany. E-mail addresses: cornelia.sindermann@uni-ulm.de (C. Sindermann), E.Duke@hud.ac.uk (&. Duke), christian.montag@uni-ulm.de (C. Montag). https://doi.org/10.1016/j.abrep.2020.100264 Received 24 October 2019; Received in revised form 22 January 2020; Accepted 10 February 2020 Available online 19 February 2020 2352-8532/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. the disordered use of the Internet in general, but to the disordered use of specific activities, such as gambling, gaming, shopping, pornography, and social networking/communication on the Internet (Brand, Young, Laier, Wölfling, & Potenza, 2016; Davis, 2001; Montag et al., 2015; Müller et al., 2017). Addictive tendencies towards SNs as a specific form of Internet Use Disorder are often referred to as “Social Network Site Addiction”, “Internet Communication Disorder”, or “Social Networks Use Disorder” (Andreassen & Pallesen, 2014; Brand et al., 2016; Montag, Wegmann, Sariyska, Demetrovics, & Brand, 2019). The present study focuses on the use of the SN Facebook and therefore uses the term “Facebook Use Disorder” when referring to the overuse of Facebook. The term “SN Use Disorder” will be used when referring to overuse of SNs in general. We consciously use the term “Disorder” to distinguish disordered use of SNs (involving the symptoms and negative consequences outlined below) from frequent, but non-problematic, use of SNs (see also the debate surrounding the term Internet Use Disorder (e.g. Pontes, Kuss, & Griffiths, 2015)). We view putative SN/Facebook Use Disorder as the extreme pole of a continuum, ranging from no/ normal via problematic to disordered use of SNs/Facebook. Drawing on classifications of substance-use disorders, SN Use Disorder is typically discussed in terms of symptoms such as salience, tolerance, mood modification, withdrawal, conflict, and relapse, as well as problems and negative consequences in daily life (Andreassen, 2015; Griffiths, Kuss, & Demetrovics, 2014). It should be noted, however, that this adaptation of the term and symptoms of addiction to SN use is a topic of debate among researchers, e.g. whether it is appropriate to apply traditional criteria for classifying substance-use disorders to addictive behaviours, such as SN Use Disorder (Kardefelt-Winther et al., 2017). In addition to these symptoms, the identification of factors that may signal a vulnerability to the development of SN Use Disorders is of interest to researchers and clinicians. Several models and explanations of specific Internet Use Disorders exist (Andreassen, 2015; Brand et al., 2016; Davis, 2001). One prominent model is the Interaction of PersonAffect-Cognition-Execution (I-PACE) Model (Brand et al., 2016). This model proposes the interaction of personal (P), affective (A), cognitive (C), and executive (E) variables in the emergence of a specific Internet Use Disorder. Personality is one of the personal (P) factors implicated in the model and works to create a vulnerability or resilience to the development of a specific Internet Use Disorder (Brand et al., 2016). Drawing on the theoretical framework of this model, the second aim of the present study was to investigate which specific personality traits would be associated with tendencies towards Facebook Use Disorder. In the field of personality research, one of the most prominent personality models is the Five-Factor Model of personality. According to this model (e.g. (Fiske, 1949)), personality can be described on the basis of five broad factors (commonly referred to as the Big Five). These factors are typically identified as extraversion (being socially outgoing, energetic, vigorous), agreeableness (being helpful, forgiving, unselfish, considerate), conscientiousness (being thorough, orderly, not lazy, efficient), neuroticism (being downcast, tense, emotionally volatile, moody), and openness to experience (being imaginative, interested in arts, aesthetics, new ideas). Depending on the self-report measure used, these five broad factors can be further sub-divided into more precise facets via subscales. For example, the Big Five Inventory (BFI) splits each of the broad Big Five factors into two narrower sub-facets (Rammstedt & Danner, 2017). As such, extraversion is split into the two sub-facets assertiveness and activity. Assertiveness assesses how outgoing and talkative an individual is, whereas activity relates to how energetic and enthusiastic an individual is (Rammstedt & Danner, 2017; Soto & John, 2009). Facebook and other SNs have previously been investigated. Ryan and Xenos (2011) observed that Facebook users in Australia tended to report higher levels of extraversion but lower levels of conscientiousness relative to non-users. Similarly, Swiss university students who reported higher levels of extraversion and lower levels of conscientiousness were more likely to be a member of a specific SN (Wehrli, 2008). Work by Brailovskaia and Margraf (2016) compared Facebook users and nonusers in a sample of German students, but could only replicate the relationship between Facebook use and higher extraversion scores. No relationship was evident between Facebook use and conscientiousness (Brailovskaia & Margraf, 2016). This null result is also at odds with more recent work by Eşkisu, Hoşoğlu, and Rasmussen (2017), who report lower levels of conscientiousness in Facebook users compared to non-users in a sample of Turkish students. Interestingly, in this study, no significant relationship was reported between Facebook use and extraversion, contradicting previous findings (Eşkisu et al., 2017). One possible explanation for the inconsistent findings in this literature may be related to the fact that none of these studies investigated sub-facets of the Big Five. It is possible that the lack of consideration of the relationship between SN user status and facet-level traits may have resulted in suppressor effects. Such suppressor effects may account for some of the non-significant findings and, therefore, inconsistencies in the results across studies. Moreover, two of the studies used short versions of Big Five measures. Due to the brevity of such measures, it is plausible that the five factors were not fully assessed, which may also explain inconsistent findings between the studies. Finally, samples from different studies also differ in their socio-demographic makeup, e.g. mean age, male-to-female-ratio, and educational background. These differences may also, at least in part, explain the lack of consistency across the results. Despite the existence of such differences, studies rarely report or control for the possible effects of such variables. This lack of consideration of covariates underscores the need to better characterise the populations under study and to control for potential confounding effects of socio-demographic variables. 1.2.2. Social Networks Use Disorder Extant research considering the association between the broad domains of the Big Five and putative SN Use Disorder has yielded mixed findings. Wilson, Fornasier, and White (2010) found that tendencies toward SN Use Disorder were significantly negatively related to conscientiousness among a sample of Australian students, whereas a significant positive association existed with extraversion. Among a U.S. sample, significant positive correlations were found between neuroticism and a scale assessing Facebook Use Disorder (Blackwell, Leaman, Tramposch, Osborne, & Liss, 2017). In contrast to the work of Wilson et al. (2010), no significant association was observed for extraversion. Notably, agreeableness, conscientiousness, and openness were not investigated in this study (Blackwell et al., 2017). Further work investigating a Taiwanese sample, comprised predominantly of college students, revealed that agreeableness, conscientiousness, and neuroticism were significantly negatively correlated with tendencies towards Facebook Use Disorder (Tang, Chen, Yang, Chung, & Lee, 2016). This latter association stands in contrast to most of the previously mentioned findings. Most recently, a cross-national meta-analysis of findings in the area suggested that Facebook Use Disorder was positively associated with neuroticism and negatively with conscientiousness (Marino, Gini, Vieno, & Spada, 2018). The findings of this meta-analysis compliment the broader literature on addiction and addictive behaviours, which often implicates (low) conscientiousness and (high) neuroticism as vulnerability factors to the development of, e.g. substance addictions (Malouff, Thorsteinsson, Rooke, & Schutte, 2007; Terracciano, Löckenhoff, Crum, Bienvenu, & Costa, 2008), problematic Internet use (Kayiş et al., 2016; Lachmann, Duke, Sariyska, & Montag, 2019; Montag, Jurkiewicz, & Reuter, 2010), and problematic smartphone use (Lachmann et al., 2019). However, to our knowledge, no previous studies in the field of SN 1.2. Previous literature 1.2.1. Users versus non-users of social networks Putative differences in the Big Five between users and non-users of 2 Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. Use Disorder and personality have considered sub-facets of the Big Five, which would help to better characterize the association of personality and SN Use Disorder and might explain some of the heterogeneous findings across studies. The potential of socio-demographic variables such as age, male-to-female-ratio, and educational background, influencing results and potentially explaining inconsistent findings, has also rarely been studied in this context. gender, and their highest level of education. 2.3.2. Big Five Inventory (BFI) Participants also completed the German version of the Big Five Inventory (BFI) (Rammstedt & Danner, 2017). This questionnaire comprises 45 items, answered on a 5-point Likert-Scale, which ranges from 1 = “very inapplicable” to 5 = “very applicable”. The German version of the BFI contains one additional item for the agreeableness scale compared to the English language version of this questionnaire. To enable closer comparison with the English version of the measure, we omitted this additional item from our analyses. The mean scores of the five broad factors, as well as two sub-facets/subscales for each of the broad factors were calculated for each participant. The internal consistencies (assessed using Cronbach’s α) for the Big Five factors were: extraversion: α = 0.86, agreeableness: α = 0.72, conscientiousness: α = 0.82, neuroticism: α = 0.85, and openness: α = 0.79. The internal consistencies for the subscales were: extraversion: assertiveness: α = 0.83, activity: α = 0.59; agreeableness: altruism: α = 0.58, compliance: α = 0.44; conscientiousness: order: α = 0.65, self-discipline: α = 0.69; neuroticism: anxiety: α = 0.76, depression: α = 0.57; openness: aesthetics: α = 0.79, ideas: α = 0.58. Although several of the internal consistencies seem rather low, this can be accounted for by the low number of items per subscale (e.g. there are only three items on the compliance scale, which likely explains the relatively low α = 0.44). The present values also closely reflect those reported by Rammstedt and Danner (2017). Thus, we deemed the internal consistencies acceptable for the present sample. Additional information on the mean inter-item correlations for each subscale can be found in the Supplementary Material. 1.3. Aims, hypotheses, and research questions The present study followed two major aims: The first aim of the present study was to investigate differences between users and nonusers of Facebook with regard to the Big Five personality traits and their sub-facets, while taking into account potential covariates such as age, gender, and education. Secondly, we aimed to investigate the associations of the Big Five and their sub-facets with tendencies towards Facebook Use Disorder. Tests of this aim should again be controlled for the potential covariates, age, gender, and education because these are frequently overlooked in the literature and might help to reconcile some of the inconsistent findings outlined above. Based on the literature mentioned above, one could hypothesize that (i) Facebook users would score higher in extraversion and lower in conscientiousness compared to Facebook non-users, and that (ii) greater tendencies towards Facebook Use Disorder would be negatively associated with conscientiousness and positively with neuroticism. However, especially with regard to the sub-facets of the Big Five, the present study is of exploratory character. Nevertheless, we deem it as important research questions, to investigate the associations between Facebook use (versus non-use) as well as Facebook Use Disorder and the Big Five sub-facets given the reasons mentioned above. 2.3.3. Facebook use and tendencies towards Facebook Use Disorder Finally, participants answered yes/no to the question: “Do you have a Facebook account?” (answer options: “yes” (=Facebook users) and “no” (=Facebook non-users)). It was not assessed whether participants had ever had a Facebook account (i.e. and subsequently deleted it). If participants indicated that they had a Facebook account, they were asked to complete a 10-item Facebook Use Disorder scale (FUD-S). This scale was developed by our research team, based on the short version of the smartphone addiction scale (Kwon, Kim, Cho, & Yang, 2013). Thus, the term “smartphone” was replaced by “Facebook” for each item to reflect the putative Facebook Use Disorder. Based on previous work, we also reworded a small number of items to reflect the first-person perspective to make the items more clear for participants (Duke & Montag, 2017). Items were answered on a 6-point Likert-Scale ranging from 1 = “strongly disagree” to 6 = “strongly agree”. This questionnaire has previously been used and validated using a Confirmatory Factor Analysis by Sha, Sariyska, Riedl, Lachmann, and Montag (2019). A mean score across the 10 items was calculated for each participant. The internal consistency of the FUD-S was α = 0.96 in the present sample. Further information on the fit of the FUD-S in the present sample is presented in the Supplementary Material. 2. Materials and methods 2.1. Participants The study was conducted online via the SurveyCoder tool (https:// www.surveycoder.com/; https://www.ckannen.com/). We recruited participants via advertisements placed in newspapers, online, on TV, and over the radio. Whenever a researcher from our group gave an interview on smartphone, Internet, or social media use, the link to the study was presented. Participation was open to anyone who could read and write in German. Participants received feedback on their smartphone use and personality scores. This individualised feedback was used to incentivise participation in the study. Further information about the sampling procedure, exclusion criteria, and handling of outliers can be found in the Supplementary Material. After the exclusion of 60 participants, data from N = 3,835 German-speaking participants (2,366 males; 1,469 females) were analysed. The mean age of the sample was M = 32.18 (SD = 11.82) years. Ages ranged from 11 to 75 years. Of the total N = 3,835 participants, 2,629 (1,577 males; 1,052 females) reported having a Facebook account. Thus, 1,206 participants (789 males; 417 females) reported that they did not have a Facebook account. 2.4. Statistical analyses 2.2. Ethical approval and informed consent Analyses were conducted using the statistical software, R (Version 3.4.1 (R core team, 2017)), and R-Studio (Version 1.1.463 (RStudio Team, 2015)). Information on the distributions of the BFI (sub-)scales and the FUDS, as well as the associations of these variables with age, gender, and educational background, can be found in the Supplementary Material. These results prompted us to control for age and gender (but not educational background) in subsequent analyses. To investigate between group differences in personality between Facebook users and non-users, a multifactorial multivariate ANCOVA and separate multifactorial ANCOVAs were calculated. Gender and Facebook use were entered as independent variables, age was included The study was approved by the local ethics committee of Ulm University, Ulm, Germany. Informed consent was obtained electronically from all participants prior to their participation in the study. Underaged participants were asked to obtain consent from their parents/legal guardians before participating. 2.3. Self-report measures 2.3.1. Demographics Participants completed demographic questions, including their age, 3 Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. as a covariate. To correct for multiple comparisons, the alpha level was set to α = 0.05/15 = 0.0033, as a total of 15 (sub-)scales of the BFI were examined. To investigate associations between the BFI (sub-)scales and Facebook Use Disorder, Spearman partial correlations (corrected for age) between the BFI (sub-)scales and the scores in the FUD-S were calculated for the complete, male, and female sample of Facebook users. For these analyses, the alpha level was also set to α = 0.05/ 15 = 0.0033. As we did not have a directional hypothesis for each association under investigation, we tested all associations for significance in a twotailed manner. The analyses (alongside the data) are also registered at the open science framework (https://osf.io/qf2cu/). 3.2. Correlations between tendencies towards Facebook Use Disorder and personality The mean values on the FUD-S for the Facebook users sample ranged from 1 to 6 with a median of 1.30 and a mean value of M = 1.62 (SD = 0.76). As outlined in Table 3, several significant partial (corrected for age) Spearman correlations were found between the BFI (sub)scales and the FUD-S score in the group of Facebook users (n = 2,629). The strongest effects in the complete users sample, which survived Bonferroni correction for multiple comparisons (α = 0.005/ 15 = 0.0033), were observed between the FUD-S and: compliance, a sub-facet of agreeableness (rs = −0.11, p < 0.001); conscientiousness (rs = −0.12, p < 0.001) and its subscale self-discipline (rs = −0.15, p < 0.001); neuroticism (rs = 0.17, p < 0.001) and its subscales, anxiety (rs = 0.15, p < 0.001) and depression (rs = 0.17, p = 0.001); and the ideas subscale of openness (rs = −0.07, p < 0.001). Similar patterns of correlations were found for both males and females after splitting the sample by gender. Exceptions to this pattern were: (1) the relationship between order, a subscale of conscientiousness, and the FUD-S, which remained significant only among females, after correcting for multiple comparisons (rs = −0.10, p < 0.001); and (2) the correlation between the openness subscale, ideas, and the FUDS, which was not significant for the female sub-sample. However, this latter correlation did not differ significantly between males and females (z = 1.56, p = 0.119; p-values for all other differences in the correlations between males and females > 0.143). 3. Results 3.1. Differences in personality between users and non-users of Facebook A multifactorial multivariate ANCOVA and separate multifactorial ANCOVAs, with gender and Facebook use as independent variables and age as covariate, revealed significant (p < 0.05) differences between males and females for nearly all (sub-)scales of the BFI. Only the agreeableness subscale compliance yielded non-significant findings. Females scored higher than males on all (sub-)scales, except the ideas subscale of the factor openness (males > females). All significant results survived manual adjustment for multiple comparisons (α = 0.0033). However, most effect sizes were small. Please see Table 1 for descriptive statistics and detailed results. The multifactorial multivariate ANCOVA and separate multifactorial ANCOVAs also revealed significant (p < 0.05) differences between Facebook users and non-users on several (sub-)scales of the BFI. However, when considering the manually adjusted alpha-level of 0.0033, only the results for the complete extraversion scale (users > non-users) and for conscientiousness and all its subscales (users < non-users) remained significant. Overall, effect sizes were small. Please see Table 2 for descriptive statistics and more detailed results. Figs. 1 and 2 provide a graphical illustration of these results. The multivariate effect of the interaction between Facebook use and gender on BFI scores was not significant (F(15, 3816) = 1.43, p = 0.126). Therefore, the interaction effects on each BFI (sub-)scale were not investigated further. 4. Discussion The first aim of the present study was to investigate differences between users and non-users of Facebook with regard to the Big Five personality traits, while taking into account potential covariates such as age and gender (and education). Secondly, we aimed to investigate the associations between the Big Five and tendencies towards Facebook Use Disorder. Tests of these aims again controlled for the potential covariates, age and gender (and education). Finally, all analyses included exploratory analyses of possible associations between Facebook use, Facebook Use Disorder and sub-facets of the Big Five, to better characterise the relationships between personality and Facebook use. 4.1. Differences between Facebook users and non-users in personality We found that our sample of Facebook users was typically younger, showed a lower male-to-female-ratio, and had a higher educational Table 1 Descriptive statistics for the BFI for the complete sample, for the sample split by gender and the results of multifactorial ANCOVAs (effects of gender as independent variable). Extraversion Assertiveness Activity Agreeableness Altruism Compliance Conscientiousness Order Self-discipline Neuroticism Anxiety Depression Openness Aesthetics Ideas Complete sample (N = 3,835) Males (n = 2,366) Females (n = 1,469) Results ANCOVAs M SD M SD M SD F(1, 3830) p ηp2 3.31 3.25 3.45 3.47 3.49 3.47 3.40 3.09 3.38 2.84 2.92 2.69 3.65 3.52 3.71 0.77 0.87 0.80 0.55 0.64 0.70 0.64 1.01 0.65 0.76 0.85 0.95 0.60 1.00 0.58 3.23 3.17 3.38 3.42 3.41 3.47 3.33 3.01 3.32 2.72 2.77 2.62 3.63 3.39 3.74 0.77 0.87 0.80 0.54 0.62 0.68 0.64 1.00 0.64 0.74 0.82 0.95 0.60 1.00 0.58 3.44 3.38 3.57 3.54 3.62 3.49 3.51 3.21 3.47 3.05 3.18 2.81 3.69 3.72 3.65 0.76 0.86 0.78 0.56 0.64 0.72 0.63 1.02 0.65 0.75 0.83 0.94 0.60 0.95 0.58 60.31 50.86 44.97 42.18 83.32 2.30 84.48 43.37 62.65 111.01 141.76 21.03 11.97 101.04 18.58 <0.001 <0.001 <0.001 <0.001 <0.001 0.130 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 0.016 0.013 0.012 0.011 0.021 0.001 0.022 0.011 0.016 0.028 0.036 0.005 0.003 0.026 0.005 Note. Scales assessing the broad Big Five factors are bolded. ηp2 = partial eta squared. 4 Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. Table 2 Descriptive statistics for the BFI for users and non-users of Facebook and the results of multifactorial ANCOVAs (effects of user group as independent variable). Users (n = 2,629) Extraversion Assertiveness Activity Agreeableness Altruism Compliance Conscientiousness Order Self-discipline Neuroticism Anxiety Depression Openness Aesthetics Ideas Non-users (n = 1,206) Results ANCOVAs M SD M SD F(1, 3830) p ηp2 3.33 3.27 3.46 3.48 3.51 3.48 3.36 3.02 3.34 2.88 2.96 2.72 3.65 3.51 3.70 0.78 0.88 0.80 0.56 0.64 0.70 0.64 1.01 0.64 0.77 0.86 0.95 0.60 1.01 0.58 3.26 3.21 3.42 3.44 3.45 3.47 3.48 3.22 3.45 2.77 2.84 2.62 3.66 3.54 3.72 0.76 0.86 0.79 0.53 0.62 0.71 0.64 1.02 0.65 0.74 0.83 0.93 0.59 0.98 0.58 9.64 7.02 1.50 4.67 6.09 0.00 16.76 24.15 11.50 7.12 7.89 2.11 0.01 0.06 0.04 0.002 0.008 0.220 0.031 0.014 0.999 <0.001 <0.001 <0.001 0.008 0.005 0.146 0.941 0.809 0.844 0.003 0.002 0.000 0.001 0.002 0.000 0.004 0.006 0.003 0.002 0.002 0.001 0.000 0.000 0.000 Note. Scales assessing the broad Big Five factors are bolded. ηp2 = partial eta squared. Table 3 Partial Spearman correlations between the BFI and the Facebook Use Disorder Scale for the complete sample of Facebook users and split by gender. Extraversion Assertiveness Activity Agreeableness Fig. 1. Differences between Facebook users and non-users in extraversion and its subscales (M +/− 2 SE). *p < 0.001, p < 0.01, p < 0.05, two-tailed. Please note that the possible range of the BFI (sub-)scales is 1 to 5. Altruism Compliance Conscientiousness Order Self-discipline Neuroticism Anxiety Depression Openness Aesthetics Fig. 2. Differences between Facebook users and non-users in conscientiousness and its subscales (M +/− 2 SE). p < 0.001, p < 0.01, *p < 0.05, twotailed. Please note that the possible range of the BFI (sub-)scales is 1 to 5. Ideas All users (n = 2,629) Male users (n = 1,577) Female users (n = 1,052) rs = 0.04 p = 0.054 rs = 0.05 p = 0.017 rs = −0.02 p = 0.236 rs = −0.04 p = 0.028 rs = 0.02 p = 0.238 rs = −0.11 p < 0.001 rs = −0.12 p < 0.001 rs = −0.06 p = 0.003 rs = −0.15 p < 0.001 rs = 0.17 p < 0.001 rs = 0.15 p < 0.001 rs = 0.17 p < 0.001 rs = −0.04 p = 0.045 rs = 0.02 p = 0.379 rs = −0.07 p < 0.001 rs = 0.02 p = 0.455 rs = 0.03 p = 0.288 rs = −0.03 p = 0.317 rs = −0.07 p = 0.005 rs = 0.00 p = 0.914 rs = −0.13 p < 0.001 rs = −0.12 p < 0.001 rs = −0.04 p = 0.076 rs = −0.14 p < 0.001 rs = 0.17 p < 0.001 rs=0.14 p < 0.001 rs = 0.17 p < 0.001 rs = −0.06 p = 0.013 rs = 0.00 p = 0.849 rs = −0.09 p < 0.001 rs = 0.03 p = 0.263 rs = 0.05 p = 0.140 rs = −0.05 p = 0.126 rs = −0.04 p = 0.240 rs = 0.01 p = 0.702 rs = −0.09 p = 0.002 rs = −0.17 p < 0.001 rs = −0.10 p < 0.001 rs = −0.19 p < 0.001 rs = 0.14 p < 0.001 rs = 0.11 p < 0.001 rs = 0.15 p < 0.001 rs = −0.02 p = 0.622 rs = −0.01 p = 0.722 rs = −0.03 p = 0.415 Note. Scales assessing the broad Big Five factors are bolded. This table presents data for the Facebook users sample (n = 2,629), only. All correlations are corrected for age. p-values are derived from two-tailed tests. level, relative to non-users (see Supplementary Material). Additionally, we replicated previous findings that Facebook users score higher in extraversion, but lower in conscientiousness (Brailovskaia & Margraf, 2016; Eşkisu et al., 2017; Ryan & Xenos, 2011; Wehrli, 2008), however, these findings should be interpreted with caution, given the relatively small effect sizes observed for the present sample. Nevertheless, the direction of effects is in line with our previous assumptions and, in contrast to previous studies, these results controlled for the potential confounding effects of age and gender. Interestingly, sub-facets of extraversion were differentially related to the use (or non-use) of Facebook. The assertiveness facet differed significantly between Facebook users and non-users (ηp2 = 0.002), but the activity facet did not (ηp2 < 0.001). This finding suggests that those individuals who particularly like to communicate with others are more likely to use Facebook (Rammstedt & Danner, 2017). At this point it is important to note that the assertiveness facet of extraversion includes items such as “I see myself as someone who is talkative”, “I see myself as someone who is reserved” (latter one reversed scored) (Rammstedt & Danner, 2017). Hence, the present results support previous work by Kujath (2011) and Raacke and Bonds-Raacke (2008), who argue that staying in touch with friends and acquaintances is one motivation for using SNs. Accordingly, as Kujath (2011) concludes, the use of SNs like Facebook may be seen as an extension of offline social interactions. We 5 Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. urge caution in this interpretation, however, as only the user-group difference for extraversion as a whole – not for the individual sub-facets – survived the correction for multiple comparisons in the current sample. Regarding conscientiousness, Facebook non-users scored higher on all conscientiousness (sub-)scales and these effects remained significant after Bonferroni correction. Larger effects were found for the order facet compared to self-discipline (ηp2 = 0.006 vs. ηp2 = 0.003). This suggests that, relative to Facebook users, non-users are more orderly, carry out their jobs and duties more carefully and reliably (Rammstedt & Danner, 2017). One possible explanation for these differences is that conscientious individuals want to prevent the possibility of SNs negatively affecting their productivity, e.g. at work or school. Therefore, they may prefer not to use SNs like Facebook. This interpretation is supported by earlier work, suggesting that SNs were viewed as too time consuming by teenagers who chose not to use them (Baker & White, 2010). Despite the relatively small effect sizes observed, these results have important implications for future research. An increasing amount of research is conducted via SNs like Facebook and dedicated online research participation platforms. The socio-demographic and personality differences between users and non-users of Facebook highlighted by the present study indicate that samples drawn from the population of Facebook users may not be representative of the general population, which also includes non-users of Facebook. Hence, the generalisability of results derived from samples of Facebook-users is questionable. Consideration of these differences is particularly important given the growing number of studies aiming to investigate digital footprint data from SNs via Psychoinformatics methods (Insel, 2017; Montag, Duke, & Markowetz, 2016). Finally, although the effect sizes observed for the differences between users and non-users of Facebook are rather small in the present study, knowledge about the specific characteristics of Facebook users is heavily monetised. This is particularly important given the increased interest in mass persuasion (e.g. personalised advertising) tailored to personality information gleaned from digital footprints (Matz, Kosinski, Nave, & Stillwell, 2017). Given the vast popularity of Facebook (We Are Social, Hootsuite, & DataReportal, 2019), even such small effects may have important implications. Thus, the present findings are important in refining our understanding of the populations targeted by such approaches. findings remained significant after correction for multiple comparisons, among both males and females. Again, the results were controlled for the potential confounding effect of age. As the correlations with the Facebook Use Disorder scale were similarly sized for both facets of neuroticism, we cannot offer a more nuanced interpretation of the relationship between neuroticism and tendencies towards Facebook Use Disorder. Interestingly, a recent study by Peterka-Bonetta, Sindermann, Sha, Zhou, and Montag (2019) reported a relationship, in which depressive symptoms (measured with the Beck Depression Inventory – II (Beck, Steer, & Brown, 1996)) were significantly positively associated with tendencies towards Internet Use Disorder. These results underline the importance of the present findings with respect to the depression subscale of neuroticism. As mentioned in the introduction, both (higher) neuroticism and (lower) conscientiousness are also predictors of other kinds of addictions and addictive behaviours, such as (unspecified) Internet Use Disorder (Kayiş et al., 2016; Lachmann et al., 2019; Malouff et al., 2007; Montag et al., 2010, 2011; Terracciano et al., 2008). Therefore, the present study lends some support to the interpretation of Facebook Use Disorder as having an addictive nature and suggests it may be viewed as a specific Internet Use Disorder. The putatively addictive nature of Facebook has also been investigated in the realm of app-design: Certain elements of Facebook and other social media platforms seem to have been designed to prolong online time and to elicit fear of missing out (Montag, Lachmann et al., 2019). These arguments also strengthen the necessity to discuss nomenclature and symptoms related to a potential diagnosis of SN Use Disorder in future revisions of the International Classification of Diseases and the Diagnostic and Statistical Manual of Mental Disorders. However, more evidence is necessary to inform this discussion. It is also important not to over-interpret the present findings, as the effect sizes of the correlations with neuroticism and conscientiousness were quite small and direct comparisons with other addictive behaviours were not made. Finally, the negative associations of Facebook Use Disorder with compliance (a subscale of agreeableness) and ideas (a subscale of openness) survived Bonferroni correction for multiple testing. These results suggest that participants who criticise others and tend to argue with others (low scores in the compliance subscale (Rammstedt & Danner, 2017)) show higher tendencies towards Facebook Use Disorder. Potentially, social interactions involving disagreements are easier to handle in online, relative to offline, environments. This relative ease of interaction may in turn reinforce the use of SNs like Facebook, ultimately leading to higher tendencies towards the disordered use. The association with the ideas facet of openness suggests that people who are interested in different topics and new experiences (high scores in the ideas subscale (Rammstedt & Danner, 2017)) show lower tendencies towards Facebook Use Disorder. This association might be explained by the fact that individuals scoring high in the subscale ideas might strive for new experiences offline rather than spending a lot of time in front of a computer/smartphone on SNs. Alternatively, work by Marshall, Lefinghausen and Ferenczi (2015) suggests that people higher in openness may have different motivations for using Facebook, e.g. for sharing information on intellectual topics, rather than purely social interactions, which may facilitate more goal-directed engagement with the SN. However, these interpretations are preliminary and the findings reported here need further investigation and replication. 4.2. Associations between Facebook Use Disorder and personality As well as characterising differences between users and non-users of Facebook, conscientiousness was also significantly negatively related to tendencies towards Facebook Use Disorder within the users sample. This is in line with previous literature (e.g. Tang et al., 2016; Wilson et al., 2010). Moreover, this association was found for males as well as females, and all results were controlled for the potential confounding effect of age. This relationship suggests that even among those who use SNs like Facebook, individuals who are more conscientious are less likely to develop tendencies towards SN Use Disorder, such as Facebook Use Disorder. Interestingly, this relationship was characterised by a stronger association with the subscale self-discipline in comparison to the subscale order (rs = −0.15 vs. rs = −0.06). As noted previously, it may be that more conscientious individuals realise that SN use may negatively influence their productivity. The relationship between tendencies toward Facebook Use Disorder and scores on the self-discipline subscale may indicate that individuals who score highly on this subscale are proficient and potentially do not want to waste time on SNs (Baker & White, 2010). Thus, such individuals may be more skilled at controlling the amount of time spent on SNs like Facebook, and in preventing possible negative influences of SNs on their lives. However, again, the effect sizes found in the present study are relatively small and thus caution should be observed when interpreting these findings. Neuroticism and its subscales, depression and anxiety, were positively related to tendencies towards Facebook Use Disorder. These 4.3. Limitations Some potential shortcomings of the present study should also be discussed. First, the generalisability of the present results may be limited to Facebook. Facebook is clearly one of the largest SNs (We Are Social, Hootsuite, & DataReportal, 2019) and is the most frequently investigated SN, as highlighted in the introduction to this paper. Recent work by Marshall, Ferenczi, Lefringhausen, Hill, and Deng (2020) points to personality differences between Facebook users and Twitter 6 Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. users, suggesting Twitter users are higher in both openness and Machiavellianism. Thus, it is advisible to replicate the present study and to consider if different personality profiles predict engagement with different SN platforms. Secondly, FUD-S scores were quite low for most of the participants in this sample (as indicated by a median of 1.30, and a mean of M = 1.62 (SD = 0.76)). This limits the variance in the data and ultimately the generalisability of these results to samples showing more severe symptoms of SN/Facebook Use Disorder. These low scores may also explain some of the rather small effect sizes found in the present study. Next, the generalisability of the present results to samples from other cultures is questionable. Our findings broadly reflect those from an Australian sample of Internet users (Ryan & Xenos, 2011). Thus, it is likely that these findings at least generalise to Western cultures. Similarly, it should be mentioned that inconsistent findings in previous studies could be due to cultural differences in the samples, as well as measurement variance in the measures used in different countries (Błachnio, Przepiorka, Senol-Durak, Durak, & Sherstyuk, 2017). Unfortunately, it was not possible to test the moderating effect of country within the present dataset and this should be a goal for future work. Next, the present sample may be biased towards individuals who are interested in smartphones and/or social media, as much of the recruitment was carried out via media related to these topics, e.g. the study was advertised whenever one of the researchers from our group gave an interview on this topic. Crucially, the variable Facebook user status did not differentiate between participants who never had a Facebook account and participants who had one in the past, but have since deleted it. It is possible that different personality characteristics also exist between these two groups. Another possible limitation is that no causal direction can be inferred based on the present data, due to the cross-sectional design used in the study. However, the Big Five are understood as rather robust and stable personality factors, which manifest in specific behavioural patterns (Costa & McCrae, 1992). Hence, it is likely that differences in the Big Five causally explain whether individuals use Facebook or not and why some individuals may develop tendencies towards Facebook Use Disorder. As mentioned previously, the I-PACE model proposes that personality factors, including low conscientiousness, are variables that may predispose individuals to develop and maintain specific kinds of Internet Use Disorders (Brand et al., 2016). As we view Facebook Use Disorder as a specific form of Internet Use Disorder, this model holds explanatory power for the present data. investigating Facebook use versus non-use or Facebook Use Disorder. Research indicates the existence of individual differences between groups of Facebook users, not limited to differences in personality. This indicates a complex interplay of many variables, which influence how individuals use Facebook. Most likely, such groups are also differentially susceptible to the development of a Facebook Use Disorder (Lo Coco et al., 2018; Moreau, Laconi, Delfour, & Chabrol, 2015). 4.4. Future work Cornelia Sindermann: Data curation, Formal analysis, Methodology, Writing - original draft, Writing - review & editing. Éilish Duke: Writing - review & editing. Christian Montag: Data curation, Methodology, Project administration, Resources, Validation, Writing review & editing. 5. Conclusions The present study builds on the existing literature by providing evidence that Facebook users report higher extraversion and lower conscientiousness scores than non-users in a large, German-speaking sample. Facebook users also differed from non-users in key socio-demographic variables, including age and male-to-female-ratio. This has crucial implications for future studies aiming to collect data from Facebook users. Additionally, we provide evidence that low conscientiousness and high neuroticism are linked to tendencies towards Facebook Use Disorder. Therefore, the present study provides further nuance to our understanding of the associations of socio-demographic variables, personality, and Facebook use. Funding This work was supported by the German Academic Scholarship Foundation (Studienstiftung des deutschen Volkes) [CS did hold a scholarship from this foundation]; the German Research Foundation (Deutsche Forschungsgemeinschaft) [grant number DFG, MO2363/32]. None of the funding sources had any influence on the study design; the collection, analysis and interpretation of the data; on writing of the manuscript; or on the decision to submit the manuscript for publication. Authors’ contributions CM planned the study design and implemented data collection. CS conducted the statistical analyses, writing of the manuscript and interpretation of the data. CM worked over the manuscript. ÉD also worked over the manuscript, gave helpful advice and improved the English language. All authors agreed upon the final version and submission of the manuscript. CRediT authorship contribution statement Future work should consider which specific functions of Facebook are driving the use of Facebook and other SNs. For example, it remains unclear whether high extraversion is associated with Facebook use solely because of the platform’s communication functionality (Kujath, 2011; Raacke & Bonds-Raacke, 2008), or if this relationship can also be explained via other Facebook features. The present data do not permit conclusions as to whether tendencies towards Facebook Use Disorder pertain to overall Facebook use or towards certain functions of Facebook. This is underlined by work by Rothen et al. (2018), who found that problematic use of Facebook was linked to preferences for specific functions. Hence, it is also unclear whether high neuroticism is linked to addictive tendencies towards all functions of Facebook or only toward specific functions. A meta-analysis by Liu and Campbell (2017) suggested that the Big Five show differential associations with the use (not tendencies towards a Use Disorder) of specific SN activities. For example, neuroticism was significantly associated with updating one’s status, but not with SN gaming, information seeking, interaction or any of the other specific activities investigated (Liu & Campbell, 2017). Hence, analysing associations between the Big Five (and their sub-facets) and specific activities carried out via the SN and/or sub-types of disordered SN use, would be an interesting avenue for future research. Finally, personality is not the only variable of interest when Declarations of Competing Interest Dr. Montag mentions that he has received (to Ulm University and earlier University of Bonn) grants from the German Research Foundation (DFG) and the German Federal Ministry for Research and Education. Dr. Montag has performed grant reviews for several agencies; has edited journal sections and articles; has given academic lectures in clinical or scientific venues or companies; and has generated books or book chapters for publishers of mental health texts. For some of these activities he received royalties, but never from the gaming or social media industry. Dr. Montag mentions that he is part of a discussion circle (Digitalität und Verantwortung: https://about.fb.com/ de/news/h/gespraechskreis-digitalitaet-und-verantwortung/) debating ethical questions linked to social media, digitalization and society/democracy at Facebook. In this context, he receives no salary for his activities. The authors declare that they have no competing interests. Finally, Dr. Montag mentions that he is currently on the scientific advisory board of the Nymphenburg group. 7 Addictive Behaviors Reports 11 (2020) 100264 C. Sindermann, et al. Acknowledgements 190–198. https://doi.org/10.1016/j.chb.2018.04.044. Malouff, J. M., Thorsteinsson, E. B., Rooke, S. E., & Schutte, N. S. (2007). Alcohol involvement and the five-factor model of personality: A meta-analysis. Journal of Drug Education, 37(3), 277–294. https://doi.org/10.2190/DE.37.3.d. 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https://openalex.org/W2566961453	https://www.epj-conferences.org/10.1051/epjconf/201713203009/pdf	English	null	Experimental study of emission Z-pinch spectra in the axial and radial directions at the Angara-5-1 facility	EPJ web of conferences	2,016	cc-by	1,241	Experimental study of emission Z-pinch spectra in the axial and radial directions at the Angara- 5-1 facility V.V. Alexandrov1, G.S. Volkov1, E.V. Grabovsky1, A.N. Gritsuk1,, K.N. Mitrofanov1 G.M. Oleinik1, and A.P. Shevelko2 V.V. Alexandrov1, G.S. Volkov1, E.V. Grabovsky1, A.N. Gritsuk1,, K.N. Mitrofanov1 G.M. Oleinik1, and A.P. Shevelko2 1State Research Center of the Russian Federation Troitsk Institute for Innovation and Fusion Research, 108840 Troitsk, Moscow, Russia 2 P.N. Lebedev Physical Institute RAS, 119991 Moscow, Russia 1State Research Center of the Russian Federation Troitsk Institute for Innovation and Fusion Research, 108840 Troitsk, Moscow, Russia 2 P.N. Lebedev Physical Institute RAS, 119991 Moscow, Russia Abstract. Data on the energy, power and spectra composition of the soft x-ray pulse of powerful Z-pinch plasmas in the axial and radial directions in the photon energy range of 0.02 - 2 keV are presented. The data are obtained from the analysis of experimental results on the implosion of cylindrical arrays with a diameter of 1.2 cm and a height of 1.6 cm of tungsten wires diameter of 6 μm, the linear mass of 220 μg/cm at a current in the range of 2.2 – 3.5 MA at the Angara-5-1 facility. Currently, to create a substance of high energy density using a spatial concentration of energy flows generated by powerful sources of energy - energy drivers. One of these drivers is a powerful source of soft X-ray radiation (SXR) on the basis of Z-pinch discharges with the use of multi-module accelerators. The spatial distribution of the plasma density during the process of implosion stagnation Z-pinch has a significant impact on the current distribution, the dissipation of magnetic energy, the duration and power of soft X- rays. Implosion of multiwire array starts at the local depletion of the material, so it is not uniform in the axial direction and in the azimuth plane. The experiments were performed at the Angara-5-1 facility, which consists of eight parallel modules. The amplitude of the pulse current reaches 3.5 MA, maximum voltage at the beginning of the water transmission line is 0.8–0.9MV, voltage pulse width at half maximum of 100 ns. The power and spatial anisotropy of the pinch SXR emission were measured using two identical sets of four vacuum X-ray detectors (XRDs) on the basis of open vacuum photodiodes with different filters [1]. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: griar@triniti.ru , 0 (2017) EPJ Web of Conferences SPECTROSCOPY.SU 2016 132 3009 , 0 (2017) EPJ Web of Conferences SPECTROSCOPY.SU 2016 132 3009 DOI: 10.1051/ 713203009 epjconf/201 Experimental study of emission Z-pinch spectra in the axial and radial directions at the Angara- 5-1 facility XUV emission spectra of Z-pinch plasma were recorded using the grazing incidence spectrograph GIS-1 (concave grating with a radius of R = 1 m, 600 grooves/mm, W/Re coating, grazing angle of 4°) in the spectral range of 20–400 Å [2]. In the experiments, the power and energy of Z-pinch radiation were obtained in different spectral ranges using XRD-detectors in the axial and radial directions. Anisotropy obtained for these directions is characteristic for all types of loads. The size of the region emitting soft X-rays was determined by this value of the radiation anisotropy. It was found , 0 (2017) EPJ Web of Conferences SPECTROSCOPY.SU 2016 132 3009 DOI: 10.1051/ 713203009 epjconf/201 that the size of the emitting region is 4–5 mm in the SXR range for cylindrical multiwire arrays at peak X-ray power, which corresponds to approximately two to threefold radial compression of the plasma. The size of the emitting region at the time of stagnation, approximately coincides in the ranges of 20-70 eV and 120-280 eV. At the same time the image obtained by the time-integrated pinhole camera indicates the presence of a considerable amount of radiating plasma surrounding the pinch – “trailing” mass [3]. that the size of the emitting region is 4–5 mm in the SXR range for cylindrical multiwire arrays at peak X-ray power, which corresponds to approximately two to threefold radial compression of the plasma. The size of the emitting region at the time of stagnation, approximately coincides in the ranges of 20-70 eV and 120-280 eV. At the same time the image obtained by the time-integrated pinhole camera indicates the presence of a considerable amount of radiating plasma surrounding the pinch – “trailing” mass [3]. The spectra of the emitting plasma were obtained with a spatial resolution. Using these spectra spatial and spectral parameters of the “trailing” mass were determined. These parameters play an important role in the dynamics of implosions and energy power of the whole process because part of the current can flow through it. This could reduce plasma energy near the axis of the array, which is converted into SXR. It is shown that the presence of a trailing” mass reduces the spectral density of radiation and changes the characteristics of spectrum. Sizes of the radiation region obtained from the spectra with a radial resolution coincide with those obtained by XRD-detectors from the radiation anisotropy and pinhole camera images. Experimental study of emission Z-pinch spectra in the axial and radial directions at the Angara- 5-1 facility Comparison of experimental and simulated spectra of radiation of tungsten Z-pinch was carried out. The procedure to restore the power spectral distribution in different photon energy ranges was solved using data from two sets of XRD with various filters. It has been shown that the results of measurement of the spectra in the radial direction, obtained by independent methods (from the XRD-detectors and the GIS-1 spectrograph), coincide. In the experiment, it was found that the plasma emission of Z-pinch tungsten multiwire cylindrical array in a radial direction is more "harder" than in the axial direction. That is in a radial direction main part of energy is emitted in the range of 70 - 300 eV, while in the axial direction radiating energy is distributed more evenly between the ranges 20 - 70 and 70 - 300 eV. Thus, the distribution of the SXR Z-Pinch source intensity is relatively isotropic in its height and in the azimuthal angle, and the spectral composition of the radiation in the axial direction corresponds to a “softer” spectrum as compared to the radial. This work was supported by the Russian Science Foundation under Project № 16-12- 10487 and in part by the Russian Foundation for Basic Research under Project 15-02- 04411. 1. A.V. Branitskiy, G.M. Oleinik, Instrum. Exp. Tech. 22, 480 (2000) 2. A.P. Shevel’ko, D.E. Bliss, E.D. Kazakov, M.G. Mazarakis, J.S. McGurn, L.V. Knight, K.W. Struve, I.Yu. Tolstikhina, T.J. Weeks, Plasma Phys. Rep. 34, 944 (2008) 3. A.N. Gritsuk, V.V. Aleksandrov, E.V. Grabovskiy, Y. Laukhin, K.N. Mitrofanov, G.M. Oleinik, G.S. Volkov, I.N. Frolov, A.P. Shevel’ko, IEEE Transactions on Plasma Science 41, 3184 (2013) 1. A.V. Branitskiy, G.M. Oleinik, Instrum. Exp. Tech. 22, 480 (2000) 2. A.P. Shevel’ko, D.E. Bliss, E.D. Kazakov, M.G. Mazarakis, J.S. McGurn, L.V. Knight, K.W. Struve, I.Yu. Tolstikhina, T.J. Weeks, Plasma Phys. Rep. 34, 944 (2008) 3. A.N. Gritsuk, V.V. Aleksandrov, E.V. Grabovskiy, Y. Laukhin, K.N. Mitrofanov, G.M. Oleinik, G.S. Volkov, I.N. Frolov, A.P. Shevel’ko, IEEE Transactions on Plasma Science 41, 3184 (2013) 1. A.V. Branitskiy, G.M. Oleinik, Instrum. Exp. Tech. 22, 480 (2000) References 3. A.N. Gritsuk, V.V. Aleksandrov, E.V. Grabovskiy, Y. Laukhin, K.N. Mitrofanov, G.M. Oleinik, G.S. Volkov, I.N. Frolov, A.P. Shevel’ko, IEEE Transactions on Plasma Science 41, 3184 (2013) 2 2
https://openalex.org/W2952897997	https://europepmc.org/articles/pmc6627811?pdf=render	English	null	Overexpression of a Metallothionein 2A Gene from Date Palm Confers Abiotic Stress Tolerance to Yeast and Arabidopsis thaliana	International journal of molecular sciences	2,019	cc-by	15,929	Received: 8 April 2019; Accepted: 11 June 2019; Published: 12 June 2019 Abstract: Although the date palm tree is an extremophile with tolerance to drought and certain levels of salinity, the damage caused by extreme salt concentrations in the soil, has created a need to explore stress-responsive traits and decode their mechanisms. Metallothioneins (MTs) are low-molecular-weight cysteine-rich proteins that are known to play a role in decreasing oxidative damage during abiotic stress conditions. Our previous study identiﬁed date palm metallothionein 2A (PdMT2A) as a salt-responsive gene, which has been functionally characterized in yeast and Arabidopsis in this study. The recombinant PdMT2A protein produced in Escherichia coli showed high reactivity against the substrate 5′-dithiobis-2-nitrobenzoic acid (DTNB), implying that the protein has the property of scavenging reactive oxygen species (ROS). Heterologous overexpression of PdMT2A in yeast (Saccharomyces cerevisiae) conferred tolerance to drought, salinity and oxidative stresses. The PdMT2A gene was also overexpressed in Arabidopsis, to assess its stress protective function in planta. Compared to the wild-type control, the transgenic plants accumulated less Na+ and maintained a high K+/Na+ ratio, which could be attributed to the regulatory role of the transgene on transporters such as HKT, as demonstrated by qPCR assay. In addition, transgenic lines exhibited higher chlorophyll content, higher superoxide dismutase (SOD) activity and improved scavenging ability for reactive oxygen species (ROS), coupled with a better survival rate during salt stress conditions. Similarly, the transgenic plants also displayed better drought and oxidative stress tolerance. Collectively, both in vitro and in planta studies revealed a role for PdMT2A in salt, drought, and oxidative stress tolerance. Keywords: metallothionein; abiotic stress; date palm; salinity; drought; functional characterization Overexpression of a Metallothionein 2A Gene from Date Palm Confers Abiotic Stress Tolerance to Yeast and Arabidopsis thaliana 1 Department of Biology, College of Sciences, Sultan Qaboos University, P.O. Box 36, 123 Muscat, Oman; himanshu30@gmail.com (H.V.P.); i.alharrasi@gmail.com (I.A.-H.); latifakharusi@gmail.com (L.A.K.); gerryjana@gmail.com (G.A.J.) 1 Department of Biology, College of Sciences, Sultan Qaboos University, P.O. Box 36, 123 Muscat, Oman; himanshu30@gmail.com (H.V.P.); i.alharrasi@gmail.com (I.A.-H.); latifakharusi@gmail.com (L.A.K.); gerryjana@gmail.com (G.A.J.) 1 Department of Biology, College of Sciences, Sultan Qaboos University, P.O. Box 36, 123 Muscat, Oman; himanshu30@gmail.com (H.V.P.); i.alharrasi@gmail.com (I.A.-H.); latifakharusi@gmail.com (L.A.K.); gerryjana@gmail.com (G.A.J.) g yj g 2 Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, P.O. Box 34, 123 Muscat, Oman; alyahyai@squ.edu.om 2 Department of Crop Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, PO Box 34 123 Muscat Oman; alyahyai@squ edu om y y q 3 Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078, USA ramanjulu.sunkar@okstate.edu * Correspondence: myaish@squ.edu.om; Tel.: +968-2414-6823 International Journal of Molecular Sciences International Journal of Molecular Sciences www.mdpi.com/journal/ijms 1. Introduction Abiotic stresses are rapidly aﬀecting agricultural lands, constantly diminishing the productivity and quality of agricultural crops [1,2]. The global population is escalating and the demand for food production is rapidly increasing [3]. In order to provide a solution for sustainable agriculture, improving the ability of plants to tolerate abiotic stresses is necessary [4]. Date palm (Phoenix dactylifera L.) is an economically important fruit tree and has a long history of cultivation in the Middle East and Northern Africa [5]. Date palm cultivation is a major component of Oman’s agriculture and the country Int. J. Mol. Sci. 2019, 20, 2871; doi:10.3390/ijms20122871 www.mdpi.com/journal/ijms www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 2871 2 of 21 is amongst the top 10 date palm producers in the world [6]. In recent years, date palm cultivation has been markedly aﬀected by invading abiotic stresses, including salinity, and therefore improving the plant’s stress tolerance is a necessity [7,8]. Overuse of groundwater reserves, lack of rainfall and irrigation with brackish water are the main causes of drought and salinity in this region [9,10]. The date palm plant is an extremophile with tolerance to various abiotic stresses such as heat, drought and salinity; however, lately the productivity and quality of fruits has been aﬀected by these stress conditions [7,11]. Studies on the response of date palm to abiotic stresses are limited and the explicit mechanisms of stress tolerance are yet to be identiﬁed. The transcriptome, methylome and miRNAome of salinity-stressed date palm oﬀer some insights into the complex abiotic stress tolerance mechanisms [12–17]. Speciﬁcally, the global gene expression proﬁles of salt-stressed date palm have identiﬁed a diﬀerentially expressed genetic makeup [12]. Along the same lines, a yeast functional bioassay aimed at identifying salt-stress-responsive genes has identiﬁed several genes, including a metallothionein (MT) gene [18]. g MTs are low-molecular-weight cysteine-rich proteins, well known for their role in the sequestration of heavy metals such as cadmium and mercury. In addition, MTs play an important role in regulation of homeostasis of essential metals such as zinc and copper [19,20]. In addition to sequestration of heavy metals, the cysteine residues of the MTs are directly and indirectly involved in the elimination of reactive oxygen species (ROS), which tend to accumulate to toxic levels during abiotic and biotic stress conditions [21–25]. MTs are present in all plants, animals and fungi and also in some prokaryotic organisms [19]. 1. Introduction MTs are classiﬁed into 15 families, based on their taxonomic features and the distribution of the cysteine residues [20]. MTs in plants belong to family 15 and are further divided into four types (i.e., MT1 (12 Cys), MT2 (14 Cys), MT3 (10 Cys) and MT4 (17 Cys) [21]). Several studies have previously shown that MTs play a role in conditions of drought and salinity [24,26–30]. In addition, the expression of MTs is regulated by various factors, including environmental stimuli, pathogens, wounds, cold, heat stress and hormone treatment [31,32]. For instance, transgenic tobacco overexpressing the GhMT3a gene of Gossypium hirsutum L., displayed improved tolerance of cold, drought and salinity. Similarly, transgenic Arabidopsis expressing the ZjMT gene of Ziziphus jujuba exhibited enhanced salinity tolerance during germination [33,34]. Additionally, MTs are important players in a variety of physiological processes, such as seed germination, root development and fruit ripening [35–37]. Furthermore, MTs are widely considered to be a component of the antioxidant defense system [38] and could also be involved in the modulation of the basic transcription process by regulating the binding to DNA of zinc ﬁnger peptides [39]. In this study, an attempt was made to functionally characterize date palm’s salt-stress-inducible metallothionein 2A (PdMT2A) gene in yeast and transgenic Arabidopsis thaliana. The aim of the study was to determine the ability of PdMT2A to enhance abiotic stress tolerance, especially under drought and salinity stress conditions. The results revealed that the transgenic yeast strain was more tolerant to drought, salinity and oxidative stresses. Similarly, the transgenic Arabidopsis plants overexpressing PdMT2A exhibited improved tolerance when exposed to drought, salinity and oxidative stress conditions. The results obtained from this study may provide valuable information relating to the abiotic stress tolerance mechanisms in date palm. Determination of the function of genes involved in common abiotic tolerance mechanisms, such as MTs, is a prerequisite for identiﬁcation of novel mechanisms in date palm, as a model abiotic-stress-tolerant fruit plant. 2.1. Sequence Analysis Revealed the Presence of Common Metallothionein Fingerprints for PdMT2 Computational analysis revealed that PdMT2A (GenBank accession XM_008804521.2) is a 234-bp sequence, coding for a 77-amino-acid-long polypeptide with a molecular weight of 7.62 kDa and a theoretical isoelectric point (pI) of 4.38. The phylogenetic analysis indicated that the Phoenix dactylifera L. metallothionein 2A is closely related to that of Elaeis guineensis (African oil palm), followed by 3 of 21 Int. J. Mol. Sci. 2019, 20, 2871 Zea mays L. (maize) and Musa acuminate (banana), which are clustered within the same clade (Figure 1A). The PdMT2A protein sequence analysis showed the presence of 14 cysteine residues which are located within the cysteine-rich domain, one each at the N- and C-termini, which is consistent with metallothionein proteins from other plant species (Figure 1B). The mean hydrophobicity plot, according to the Kyte–Doolittle scale, showed that a large portion of the protein is hydrophilic (Figure 1C), which is also consistent with other previously characterized metallothioneins of the same class from other plant species. In order to predict the expression behavior of PdMT2A under diﬀerent environmental conditions, the putative promoter sequence upstream of the start codon was computationally analyzed. The analysis revealed that the putative promoter consisted of 32% abiotic-stress-responsive transcription factor binding sites (TFBSs), amongst the total cis-regulatory elements analyzed in the sequence. The majority of TFBSs include myeloblastosis viral oncogene (MYB), apetala2/ethylene response factor (AP2/ERF) and basic domain-leucine zipper (bZIP) cis-regulatory elements, amongst which most of the AP2/ERF and MYB regulatory elements were located close to the start codon (Figure 1D). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 3 of 21 clade (Figure 1A). The PdMT2A protein sequence analysis showed the presence of 14 cysteine residues which are located within the cysteine-rich domain, one each at the N- and C-termini, which is consistent with metallothionein proteins from other plant species (Figure 1B). The mean hydrophobicity plot, according to the Kyte–Doolittle scale, showed that a large portion of the protein is hydrophilic (Figure 1C), which is also consistent with other previously characterized metallothioneins of the same class from other plant species. In order to predict the expression behavior of PdMT2A under different environmental conditions, the putative promoter sequence upstream of the start codon was computationally analyzed. The analysis revealed that the putative promoter consisted of 32% abiotic-stress-responsive transcription factor binding sites (TFBSs), amongst the total cis-regulatory elements analyzed in the sequence. 2.1. Sequence Analysis Revealed the Presence of Common Metallothionein Fingerprints for PdMT2 The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid diﬀerence. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative promoter sequence (D). 2.1. Sequence Analysis Revealed the Presence of Common Metallothionein Fingerprints for PdMT2 The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid difference. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative Figure 1. Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid diﬀerence. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative promoter sequence (D). Figure 1. Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid difference. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative Figure 1. Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). 2.1. Sequence Analysis Revealed the Presence of Common Metallothionein Fingerprints for PdMT2 The majority of TFBSs include myeloblastosis viral oncogene (MYB), apetala2/ethylene response factor (AP2/ERF) and basic domain-leucine zipper (bZIP) cis-regulatory elements, amongst which most of the AP2/ERF and MYB regulatory elements were located close to the start codon (Figure 1D) gulatory elements were located close to the start codon (Figure 1D). Figure 1. Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid difference. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative Figure 1. Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. The gray-colored highlighting represents identical and conserved regions and the dark highlighting represents similar regions with one amino acid diﬀerence. The two cysteine-rich domains are indicated at the N- and C-termini of the sequences (B). The hydrophobicity plot of each amino acid in the PdMT2A sequence according to the Kyte–Doolittle hydrophobicity scale (C). The sequence analysis of the putative 2000-bp promoter region upstream of the PdMT2A start codon, showing the abundance of abiotic-stress-related transcription factor binding sites (TFBSs) within the putative promoter sequence (D). Figure 1. Sequence analysis of 10 metallothioneins (MT) amino acid sequences of various plant species and the phylogenetic tree constructed using the neighbor-joining method. The bootstrap values on the nodes represent percentages of 1000 repetitions (A). The multiple sequence alignment of the deduced amino acid sequence of PdMT2A and other MT2A isoforms from other plant species. 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast f g The PdMT2A gene was cloned into yeast expression vector pYES-DEST52 (Figure 3A) and transformed into yeast cells, in order to test its function in a basic eukaryotic system. Initially, the recombinant (TY) and the empty plasmids (EV) were transformed into the wild-type yeast strain INVSc1 as a control in the yeast assays. The comparative spot growth responses between TY and EV under different abiotic stresses were assayed, but no differences were observed (Figure S1). As a result, we decided to use the salt-sensitive yeast strain BYT458. The yeast spot assay showed that both transgenic (TY) and empty vector (EV) cells had uniform growth under control conditions. However, the TY cells had enhanced growth on plates containing PEG (polyethylene glycol) and H2O2, suggesting the involvement of PdMT2A in drought and oxidative stress tolerance. On plates supplemented with LiCl, the EV cells showed slightly better growth than the TY cells, whereas on the plates containing 300 mM NaCl, the TY and EV cells did not show visible differences in growth pattern (Figure 3B). The pattern of yeast growth was also tested in liquid synthetic medium (LSM) supplemented with 50 mM NaCl. NaCl concentrations of 300 mM and 50 mM were selected for these assays because they were the highest sustainable concentrations for this strain of yeast when grown on solid and liquid media, respectively. The TY cells showed a high growth rate during the lag and exponential growth phases, compared to the EV cells (Figure 3C). Concentrations of LiCl and H2O2 as small as 1 mM and 0.5 mM, respectively, were detrimental for yeast in liquid culture. However, sorbitol concentration as high as 1 M did not lead to differences in the growth patterns, and therefore further liquid culture assays were not performed for these stresses. The PdMT2A gene was cloned into yeast expression vector pYES-DEST52 (Figure 3A) and transformed into yeast cells, in order to test its function in a basic eukaryotic system. Initially, the recombinant (TY) and the empty plasmids (EV) were transformed into the wild-type yeast strain INVSc1 as a control in the yeast assays. The comparative spot growth responses between TY and EV under diﬀerent abiotic stresses were assayed, but no diﬀerences were observed (Figure S1). As a result, we decided to use the salt-sensitive yeast strain BYT458. p promoter sequence (D). 2.2. Reactivity of PdMT2A Protein Against DTNB In Vitro 2.2. Reactivity of PdMT2A Protein Against DTNB In Vitro In order to determine the molecular function of PdMT2A, the PdMT2A gene was cloned into pTYB21 plasmid (Figure 2A), overexpressed in Escherichia coli and purified using affinity chromatography. However, due to the small size and the instability of the protein, partially purified recombinant PdMT2A protein was obtained. The reactivity of the PdMT2A recombinant protein with 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) was tested, against the proteins from cells harboring a pTYB21 empty vector as a negative control (Figure 2B). This assay showed that the PdMT2A protein hibit d ti it b t t i hi h th ti t l Th ti f ffi it h d In order to determine the molecular function of PdMT2A, the PdMT2A gene was cloned into pTYB21 plasmid (Figure 2A), overexpressed in Escherichia coli and puriﬁed using aﬃnity chromatography. However, due to the small size and the instability of the protein, partially puriﬁed recombinant PdMT2A protein was obtained. The reactivity of the PdMT2A recombinant protein with 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) was tested, against the proteins from cells harboring a pTYB21 empty vector as a negative control (Figure 2B). This assay showed that the PdMT2A protein exhibited a reactivity about twice as high as the negative control. The time course of aﬃnity showed that the PdMT2A protein had a high initial reaction speed for the reaction with DTNB (Figure 2C). 4 of 21 4 of 21 Int. J. Mol. Sci. 2019, 20, 2871 Int J Mol Sci 2018 19 x FOR Figure 2. Production of recombinant PdMT2A protein in E. coli. Schematic representation of the expression vector (A). The polyacrylamide gel image of the partially purified recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C). Figure 2. Production of recombinant PdMT2A protein in E. coli. Schematic representation of the expression vector (A). The polyacrylamide gel image of the partially puriﬁed recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C). Figure 2. p promoter sequence (D). 2.2. Reactivity of PdMT2A Protein Against DTNB In Vitro Production of recombinant PdMT2A protein in E. coli. Schematic representation of the expression vector (A). The polyacrylamide gel image of the partially purified recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C). Figure 2. Production of recombinant PdMT2A protein in E. coli. Schematic representation of the expression vector (A). The polyacrylamide gel image of the partially puriﬁed recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C). expression vector (A). The polyacrylamide gel image of the partially purified recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5’-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C). expression vector (A). The polyacrylamide gel image of the partially puriﬁed recombinant PdMT2A protein and the protein produced by the pTYB21 empty vector in E. coli. (B). The reactivity of the recombinant PdMT2A protein and the empty vector against the 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) substrate shows the initial speed of the reaction from the plot of absorbance versus time (C). 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast The yeast spot assay showed that both transgenic (TY) and empty vector (EV) cells had uniform growth under control conditions. However, the TY cells had enhanced growth on plates containing PEG (polyethylene glycol) and H2O2, suggesting the involvement of PdMT2A in drought and oxidative stress tolerance. On plates supplemented with LiCl, the EV cells showed slightly better growth than the TY cells, whereas on the plates containing 300 mM NaCl, the TY and EV cells did not show visible diﬀerences in growth pattern (Figure 3B). The pattern of yeast growth was also tested in liquid synthetic medium (LSM) supplemented with 50 mM NaCl. NaCl concentrations of 300 mM and 50 mM were selected for these assays because they were the highest sustainable concentrations for this strain of yeast when grown on solid and liquid media, respectively. The TY cells showed a high growth rate during the lag and exponential growth phases, compared to the EV cells (Figure 3C). Concentrations of LiCl and H2O2 as small as 1 mM and 0.5 mM, respectively, were detrimental for yeast in liquid culture. However, sorbitol concentration as high as 1 M did not lead to diﬀerences in the growth patterns, and therefore further liquid culture assays were not performed for these stresses. further liquid culture assays were not performed for these stresses. The effect of PdMT2A on the Na+ and K+ balance was tested by measuring Na+ and K+ concentrations in the TY and EV cells grown in LSM supplemented with 25 mM NaCl. Although Na+ and K+ concentrations were elevated under salt stress, no significant differences were observed between TY and EV cells (Figure 3D). The eﬀect of PdMT2A on the Na+ and K+ balance was tested by measuring Na+ and K+ concentrations in the TY and EV cells grown in LSM supplemented with 25 mM NaCl. Although Na+ and K+ concentrations were elevated under salt stress, no signiﬁcant diﬀerences were observed between TY and EV cells (Figure 3D). 5 of 21 5 of 21 Int. J. Mol. Sci. 2019, 20, 2871 Int. J. Mol. Sci. 2018, 19, x FOR Figure 3. Overexpression of PdMT2A in yeast. Schematic representation of the cloned PdMT2A gene within the pYES-DEST52 plasmid (A). 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast The effect of the PdMT2A transgene on the growth of yeast cells and the relative tolerance of transgenic (TY) and empty vector (EV) cells tested by yeast spot assay when grown under control and various abiotic stress conditions on solid media (B). Yeast liquid culture assay used to test the relative tolerance of TY and EV cells grown under control and 50 mM NaCl stress conditions (C). Accumulation of Na+ and K+ in TY or EV cells when grown under control and 25 mM NaCl salinity stress conditions (D). The bars represent the mean concentration of Na+ and K+ (± SE, n = 3). Figure 3. Overexpression of PdMT2A in yeast. Schematic representation of the cloned PdMT2A gene within the pYES-DEST52 plasmid (A). The eﬀect of the PdMT2A transgene on the growth of yeast cells and the relative tolerance of transgenic (TY) and empty vector (EV) cells tested by yeast spot assay when grown under control and various abiotic stress conditions on solid media (B). Yeast liquid culture assay used to test the relative tolerance of TY and EV cells grown under control and 50 mM NaCl stress conditions (C). Accumulation of Na+ and K+ in TY or EV cells when grown under control and 25 mM NaCl salinity stress conditions (D). The bars represent the mean concentration of Na+ and K+ (± SE, n = 3). Figure 3. Overexpression of PdMT2A in yeast. Schematic representation of the cloned PdMT2A gene within the pYES-DEST52 plasmid (A). The effect of the PdMT2A transgene on the growth of yeast cells and the relative tolerance of transgenic (TY) and empty vector (EV) cells tested by yeast spot assay when grown under control and various abiotic stress conditions on solid media (B). Yeast liquid culture assay used to test the relative tolerance of TY and EV cells grown under control and 50 mM NaCl stress conditions (C). Accumulation of Na+ and K+ in TY or EV cells when grown under control and 25 mM NaCl salinity stress conditions (D). The bars represent the mean concentration of Na+ and K+ (± SE, n = 3). Figure 3. Overexpression of PdMT2A in yeast. Schematic representation of the cloned PdMT2A gene within the pYES-DEST52 plasmid (A). 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast 2.3. PdMT2A Confers Drought and Oxidative Stress Tolerance in Yeast The eﬀect of the PdMT2A transgene on the growth of yeast cells and the relative tolerance of transgenic (TY) and empty vector (EV) cells tested by yeast spot assay when grown under control and various abiotic stress conditions on solid media (B). Yeast liquid culture assay used to test the relative tolerance of TY and EV cells grown under control and 50 mM NaCl stress conditions (C). Accumulation of Na+ and K+ in TY or EV cells when grown under control and 25 mM NaCl salinity stress conditions (D). The bars represent the mean concentration of Na+ and K+ (± SE, n = 3). 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc Dot-blot immunoassay using total protein ly ed from the transgenic and the wild-type (WT) Arabidopsis plants, to demonstrate logous expression of Myc-PdMT2A protein and the actin protein using anti-Myc and anti-a Figure 4. Overexpression of PdMT2A in Arabidopsis. Schematic representation of the cloned PdMT2A gene within the pEarleyGate 203 plasmid (A). Dot-blot immunoassay using total protein lysate extracted from the transgenic and the wild-type (WT) Arabidopsis plants, to demonstrate the heterologous expression of Myc-PdMT2A protein and the actin protein, using anti-Myc and anti-actin antibodies, respectively (B). tibodies, respectively (B). he abiotic stress tolerance of the transgenic Arabidopsis seedlings was assayed on plain h Murashige and Skoog (MS) agar medium containing plates, or on MS agar p mented with NaCl, mannitol or H2O2, representing salinity, drought and oxidative stre tively. The three independent transgenic lines grown on NaCl, mannitol and H2O2 plates cantly (p < 0.05) longer roots, larger-sized leaves and greater biomass accumulation, comp respective WT plants (Figure 5). Unlike other stress conditions, where only the elongati in root was observed, a unique pattern of short main roots coupled with heavy branchi t system was observed under H2O2 stress (Figure 5D). The dry mass accumulation pattern The abiotic stress tolerance of the transgenic Arabidopsis seedlings was assayed on plain half-strength Murashige and Skoog (MS) agar medium containing plates, or on MS agar plates supplemented with NaCl, mannitol or H2O2, representing salinity, drought and oxidative stresses, respectively. The three independent transgenic lines grown on NaCl, mannitol and H2O2 plates had signiﬁcantly (p < 0.05) longer roots, larger-sized leaves and greater biomass accumulation, compared to the respective WT plants (Figure 5). Unlike other stress conditions, where only the elongation of the main root was observed, a unique pattern of short main roots coupled with heavy branching of the root system was observed under H2O2 stress (Figure 5D). The dry mass accumulation pattern was relatively greater in transgenic Arabidopsis lines subjected to drought and H2O2 treatment, compared to those under salt stress. This observation is consistent with the yeast analysis, where the transgenic yeast also displayed a better tolerance under drought and oxidative stresses compared to salt stress. y ( g ) y p ely greater in transgenic Arabidopsis lines subjected to drought and H2O2 treatment, comp e under salt stress. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc p g p g p g y The PdMT2A gene was cloned downstream of the Myc epitope tag of the binary vector (pEarleyGate 203) and transformed into Arabidopsis using the Agrobacterium-mediated transformation method (Figure 4A). The presence of the transgene in the transgenic lines was verified using PCR. In addition, a dot-blot immunoassay was performed to detect the accumulation of the heterologous PdMT2A protein in the transgenic plants, using anti-Myc antibodies. The immunoassay results detected a strong signal in the transgenic Arabidopsis lines but none in the wild-type (WT) Arabidopsis The PdMT2A gene was cloned downstream of the Myc epitope tag of the binary vector (pEarleyGate 203) and transformed into Arabidopsis using the Agrobacterium-mediated transformation method (Figure 4A). The presence of the transgene in the transgenic lines was veriﬁed using PCR. In addition, a dot-blot immunoassay was performed to detect the accumulation of the heterologous PdMT2A protein in the transgenic plants, using anti-Myc antibodies. The immunoassay results detected a strong signal in the transgenic Arabidopsis lines but none in the wild-type (WT) Arabidopsis (Figure 4B). 6 of 21 Int. J. Mol. Sci. 2019, 20, 2871 A strong signal was detected for the actin protein in WT and transgenic lines using anti-actin antibodies, which served as a loading control (Figure 4B). These results conﬁrmed the expression of PdMT2A protein in transgenic plants. ol. Sci. 2018, 19, x FOR PEER REVIEW A strong signal was detected for the actin protein in WT and transgenic lines using anti-actin antibodies, which served as a loading control (Figure 4B). These results conﬁrmed the expression of PdMT2A protein in transgenic plants. ol. Sci. 2018, 19, x FOR PEER REVIEW 6 e 4. Overexpression of PdMT2A in Arabidopsis. Schematic representation of the cloned PdMT within the pEarleyGate 203 plasmid (A). Dot-blot immunoassay using total protein lys ted from the transgenic and the wild-type (WT) Arabidopsis plants, to demonstrate t ologous expression of Myc-PdMT2A protein and the actin protein, using anti-Myc and anti-ac Figure 4. Overexpression of PdMT2A in Arabidopsis. Schematic representation of the cloned PdMT2A gene within the pEarleyGate 203 plasmid (A). Dot-blot immunoassay using total protein lysate extracted from the transgenic and the wild-type (WT) Arabidopsis plants, to demonstrate the heterologous expression of Myc-PdMT2A protein and the actin protein, using anti-Myc and anti-actin antibodies, respectively (B). 4. Overexpression of PdMT2A in Arabidopsis. Schematic representation of the cloned PdM within the pEarleyGate 203 plasmid (A). 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc This observation is consistent with the yeast analysis, where the transg lso displayed a better tolerance under drought and oxidative stresses compared to salt st y p y g p The Na+ and K+ concentrations in the stressed and non-stressed transgenic and wild-type seedlings were measured to determine the eﬀect of the transgene on Na+ and K+ homeostasis during abiotic stress conditions. As expected, Na+ concentration did not vary between transgenic and WT seedlings under control and drought conditions. On the other hand, the Na+ concentration of transgenic lines was signiﬁcantly (p < 0.05) lower than that of the WT plants under salinity (Figure 6A). Interestingly, the transgenic lines had a higher K+ concentration than the WT plants under control conditions (Figure 6B). Furthermore, under both salinity and drought conditions, the transgenic lines tended to accumulate more K+ during salt stress than the WT lines (Figure 6B). This was further evident from the K+/Na+ ratio analysis. The K+/Na+ ratio in three independent transgenic lines was signiﬁcantly (p < 0.05) higher than in the corresponding WT plants under salinity. Albeit at slightly lower levels, a similar trend of higher K+/Na+ ratio was also observed under both control and drought stress conditions in transgenic lines, compared to WT plants (Figure 6C). 7 of 21 Int. J. Mol. Sci. 2019, 20, 2871 ol. Sci. 2018, 19, x FOR PEER e 5. The effect of PdMT2A on the phenotype of the transgenic Arabidopsis seedlings. The gro n, root length and dry weight of the WT and PdMT2A transgenic Arabidopsis lines ured when the seedlings were grown on plain half-MS plates as a control (A) or on hal supplied with 100 mM NaCl (B), 150 mM mannitol (C) or 2 mM H2O2 (D), for 14 days of the main root was measured manually using a standard centimeter scale. The bars repre the mean root length in cm or the dry weight in mg (± SE, n = 3), while the asterisks indic Figure 5. The eﬀect of PdMT2A on the phenotype of the transgenic Arabidopsis seedlings. The growth pattern, root length and dry weight of the WT and PdMT2A transgenic Arabidopsis lines were measured when the seedlings were grown on plain half-MS plates as a control (A) or on half-MS plates supplied with 100 mM NaCl (B), 150 mM mannitol (C) or 2 mM H2O2 (D), for 14 days. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc The length of the main root was measured manually using a standard centimeter scale. The bars represent either the mean root length in cm or the dry weight in mg (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from WT plants (p < 0.05). 5. The effect of PdMT2A on the phenotype of the transgenic Arabidopsis seedlings. The gr n, root length and dry weight of the WT and PdMT2A transgenic Arabidopsis lines red when the seedlings were grown on plain half-MS plates as a control (A) or on hal supplied with 100 mM NaCl (B), 150 mM mannitol (C) or 2 mM H2O2 (D), for 14 days of the main root was measured manually using a standard centimeter scale. The bars repr the mean root length in cm or the dry weight in mg (± SE, n = 3), while the asterisks indic Figure 5. The eﬀect of PdMT2A on the phenotype of the transgenic Arabidopsis seedlings. The growth pattern, root length and dry weight of the WT and PdMT2A transgenic Arabidopsis lines were measured when the seedlings were grown on plain half-MS plates as a control (A) or on half-MS plates supplied with 100 mM NaCl (B), 150 mM mannitol (C) or 2 mM H2O2 (D), for 14 days. The length of the main root was measured manually using a standard centimeter scale. The bars represent either the mean root length in cm or the dry weight in mg (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from WT plants (p < 0.05). gnificant difference from WT plants (p < 0.05). he Na+ and K+ concentrations in the stressed and non-stressed transgenic and wild gs were measured to determine the effect of the transgene on Na+ and K+ homeostasis d The eﬀect of abiotic stress on photosynthesis was determined by measuring the chlorophyll levels. The transgenic lines had signiﬁcantly (p < 0.05) higher levels of chlorophyll under salinity, compared to the WT plants. Moreover, under drought and oxidative stress conditions, chlorophyll retention was greater in transgenic lines (Figure 7A). gnificant difference from WT plants (p < 0.05). he Na+ and K+ concentrations in the stressed and non-stressed transgenic and wild gs were measured to determine the effect of the transgene on Na+ and K+ homeostasis d The eﬀect of abiotic stress on photosynthesis was determined by measuring the chlorophyll levels. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc The transgenic lines had signiﬁcantly (p < 0.05) higher levels of chlorophyll under salinity, compared to the WT plants. Moreover, under drought and oxidative stress conditions, chlorophyll retention was greater in transgenic lines (Figure 7A). g g stress conditions. As expected, Na+ concentration did not vary between transgenic an ngs under control and drought conditions. On the other hand, the Na+ concentrati enic lines was significantly (p < 0.05) lower than that of the WT plants under salinity (F nterestingly, the transgenic lines had a higher K+ concentration than the WT plants u d h d b h l d d h d The proline accumulation in transgenic lines under drought and salinity was high, although only two transgenic lines showed signiﬁcantly (p < 0.05) higher accumulation than the WT plants. Conversely, the proline concentration was low in all transgenic lines under oxidative stress, compared to the WT plants (Figure 7B). Interestingly, under control conditions, the proline concentration was signiﬁcantly lower in the transgenic plants compared to the WT plants. 8 of 21 Int. J. Mol. Sci. 2019, 20, 2871 18, 19, x FOR PEER RE e effect of PdMT2A on the accumulation of Na+ and K+ content in of Na+ (A) and K+ (B) and the K+/Na+ ratio (C) in PdMT2A transg nts when subjected to control, drought (150 mM mannitol) and salinity (1 h lf h MS di l Th b h Figure 6. The eﬀect of PdMT2A on the accumulation of Na+ and K+ content in Arabidopsis. Accumulation of Na+ (A) and K+ (B) and the K+/Na+ ratio (C) in PdMT2A transgenic and WT Arabidopsis plants when subjected to control, drought (150 mM mannitol) and salinity (100 mM NaCl) conditions on half-strength MS-medium plates. The bars represent the mean concentrations of Na+ and K+ (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from the WT plants (p < 0.05). e effect of PdMT2A on the accumulation of Na+ and K+ content i n of Na+ (A) and K+ (B) and the K+/Na+ ratio (C) in PdMT2A transg ants when subjected to control, drought (150 mM mannitol) and salinity (1 Figure 6. The eﬀect of PdMT2A on the accumulation of Na+ and K+ content in Arabidopsis. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc Accumulation of Na+ (A) and K+ (B) and the K+/Na+ ratio (C) in PdMT2A transgenic and WT Arabidopsis plants when subjected to control, drought (150 mM mannitol) and salinity (100 mM NaCl) conditions on half-strength MS-medium plates. The bars represent the mean concentrations of Na+ and K+ (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from the WT plants (p < 0.05). s on half-strength MS-medium plates. The bars represent the mean concentratio SE, n = 3), while the asterisks indicate a significant difference from the WT plants t of abiotic stress on photosynthesis was determined by measuring th ansgenic lines had significantly (p < 0.05) higher levels of chlorophyll u the WT plants. Moreover, under drought and oxidative stress condition greater in transgenic lines (Figure 7A). l l d d h d l h h Lipid peroxidation levels and superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were analyzed to determine the eﬀect of the transgene on the status of the antioxidant machinery under abiotic stress conditions. To assess the eﬀect of stress on the cell membrane status, lipid peroxidation was estimated by measuring the amount of malondialdehyde (MDA) in the samples. The MDA levels were higher in only one of the transgenic lines subjected to drought stress (Figure 7C). The SOD activity was relatively high in all transgenic lines under salinity, drought and oxidative stress conditions, although the activity was signiﬁcantly (p < 0.05) higher under both salinity and oxidative stress but not under drought (Figure 7D). The APX activity of the transgenic and WT plants was similar under control conditions. However, under drought, the transgenic Arabidopsis had signiﬁcantly Int. J. Mol. Sci. 2019, 20, 2871 9 of 21 (p < 0.05) higher APX activity than WT plants. The APX enzyme activity under salinity and oxidative stress was inconsistent among the three transgenic lines and the WT plants (Figure 7E). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 21 ss was inconsistent among the three transgenic lines and the WT plants (Figure 7E). t. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 Figure 7. The effect of PdMT2A on chlorophyll, proline and antioxidant enzymes. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc The effect of drought, salinity and oxidative stress on the chlorophyll content (A), proline concentration (B), malondialdehyde (MDA) concentration (C), superoxide dismutase (SOD) activity (D) and ascorbate peroxidase (APX) activity (E) of PdMT2A transgenic and WT Arabidopsis plants. The bars represent the mean chlorophyll, proline or MDA concentration and the SOD or APX activity (± SE, n = 3), while the asterisks indicate a significant difference from the WT plants (p < 0.05). 5. The Soil-Grown Transgenic Arabidopsis Lines Displayed Improved Stress Tolerance Figure 7. The eﬀect of PdMT2A on chlorophyll, proline and antioxidant enzymes. The eﬀect of drough salinity and oxidative stress on the chlorophyll content (A), proline concentration (B), malondialdehyd (MDA) concentration (C), superoxide dismutase (SOD) activity (D) and ascorbate peroxidase (APX activity (E) of PdMT2A transgenic and WT Arabidopsis plants. The bars represent the mean chlorophy proline or MDA concentration and the SOD or APX activity (± SE, n = 3), while the asterisks indicate signiﬁcant diﬀerence from the WT plants (p < 0.05). Figure 7. The effect of PdMT2A on chlorophyll, proline and antioxidant enzymes. The effect of drought, salinity and oxidative stress on the chlorophyll content (A), proline concentration (B), malondialdehyde (MDA) concentration (C), superoxide dismutase (SOD) activity (D) and ascorbate peroxidase (APX) activity (E) of PdMT2A transgenic and WT Arabidopsis plants. The bars represent the mean chlorophyll, proline or MDA concentration and the SOD or APX activity (± SE, n = 3), while the asterisks indicate a significant difference from the WT plants (p < 0.05). Figure 7. The eﬀect of PdMT2A on chlorophyll, proline and antioxidant enzymes. The eﬀect of drought, salinity and oxidative stress on the chlorophyll content (A), proline concentration (B), malondialdehyde (MDA) concentration (C), superoxide dismutase (SOD) activity (D) and ascorbate peroxidase (APX) activity (E) of PdMT2A transgenic and WT Arabidopsis plants. The bars represent the mean chlorophyll, proline or MDA concentration and the SOD or APX activity (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from the WT plants (p < 0.05). Int. J. Mol. Sci. 2019, 20, 2871 10 of 21 10 of 21 2.5. The Soil-Grown Transgenic Arabidopsis Lines Displayed Improved Stress Tolerance 2.5. The Soil-Grown Transgenic Arabidopsis Lines Displayed Improved Stress Tolerance The transgenic lines and the WT Arabidopsis plants were grown on soil to assess their response to drought and salinity. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc Both transgenic and WT plants showed healthy growth under control conditions; however, the three transgenic lines showed an improved tolerance to drought and salinity by the eighth day of the stress treatment, compared to WT plants. The transgenic lines continued to perform better than the WT plants under salt stress even on the 14th day of the salt-stress treatment. On the other hand, by the 14th day of drought stress, both the transgenic and WT plants were completely wilted, suggesting that the stress intensity was severe. In order to determine the recovery capacity of the transgenic and WT plants after 14 days of stress treatment, both drought- and salinity-stressed plants were allowed to recover from their respective stress treatments. A small fraction of salt-stress-treated transgenic lines were able to recover but not the WT plants. Neither the transgenic lines nor the WT plants were able to recover from the drought treatment (Figure 8). Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 21 The transgenic lines and the WT Arabidopsis plants were grown on soil to assess their response to drought and salinity. Both transgenic and WT plants showed healthy growth under control conditions; however, the three transgenic lines showed an improved tolerance to drought and salinity by the eighth day of the stress treatment, compared to WT plants. The transgenic lines continued to perform better than the WT plants under salt stress even on the 14th day of the salt-stress treatment. On the other hand, by the 14th day of drought stress, both the transgenic and WT plants were completely wilted, suggesting that the stress intensity was severe. In order to determine the recovery capacity of the transgenic and WT plants after 14 days of stress treatment, both drought- and salinity- stressed plants were allowed to recover from their respective stress treatments. A small fraction of salt-stress-treated transgenic lines were able to recover but not the WT plants. Neither the transgenic li th WT l t bl t f th d ht t t t (Fi 8) Figure 8. Performance of the PdMT2A transgenic and WT Arabidopsis plants grown on soil and subjected to salinity (200 mM NaCl) and drought stress for 14 days. Figure 8. Performance of the PdMT2A transgenic and WT Arabidopsis plants grown on soil and subjected to salinity (200 mM NaCl) and drought stress for 14 days. Figure 8. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining of WT and transgenic Arabidopsis plant leaves under salinity and drought stress conditions. of WT and transgenic Arabidopsis plant leaves under salinity and drought stress conditions. The qPCR analysis was performed to determine the effect of the PdMT2A transgene on the expression level of some other genes involved in salinity tolerance. The expression level of genes associated with salt tolerance (e.g., Na+ uptake/accumulation/extrusion), was assessed in the transgenic lines compared with WT plants under stress conditions. We determined the expression level of CHX20, SOS1, HKT1, and vacuolar Na+/H+ antiporter as well as ABA and SOD genes as they The qPCR analysis was performed to determine the eﬀect of the PdMT2A transgene on the expression level of some other genes involved in salinity tolerance. The expression level of genes associated with salt tolerance (e.g., Na+ uptake/accumulation/extrusion), was assessed in the transgenic lines compared with WT plants under stress conditions. We determined the expression level of CHX20, SOS1, HKT1, and vacuolar Na+/H+ antiporter as well as ABA and SOD genes as they are known to be involved in salinity stress tolerance mechanisms in plants. The qPCR analysis was performed to determine the effect of the PdMT2A transgene on the expression level of some other genes involved in salinity tolerance. The expression level of genes associated with salt tolerance (e.g., Na+ uptake/accumulation/extrusion), was assessed in the transgenic lines compared with WT plants under stress conditions. We determined the expression level of CHX20, SOS1, HKT1, and vacuolar Na+/H+ antiporter as well as ABA and SOD genes as they are known to be involved in salinity stress tolerance mechanisms in plants. , O , , / p A A O g y are known to be involved in salinity stress tolerance mechanisms in plants. The results showed that the expression of a putative cation/H+ exchanger (CHX20) and a vacuolar Na+/H+ antiporter gene was downregulated in transgenic lines, compared to the WT plants under drought and salinity. Similarly, the expression of salt overly sensitive 1 gene (SOS1) and ABA stress-induced gene (ABA) was downregulated under drought stress. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc Performance of the PdMT2A transgenic and WT Arabidopsis plants grown on soil and subjected to salinity (200 mM NaCl) and drought stress for 14 days. Figure 8. Performance of the PdMT2A transgenic and WT Arabidopsis plants grown on soil and subjected to salinity (200 mM NaCl) and drought stress for 14 days. The accumulation of H2O2 and superoxide anion (O2−) was detected using 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining. The leaves of WT plants displayed darker spots than the transgenic Arabidopsis lines under salinity and drought stress conditions, which clearly indicates high accumulation of H2O2 and O2− in WT plants. The transgenic lines exhibited slightly lower accumulation of H2O2 under drought and salinity as indicated by the DAB staining. Additionally, the NBT staining clearly indicates a lower accumulation of O2- in transgenic plants, compared to the WT plants. This indicates that the transgenic plants had a lower accumulation of ROS, compared to the WT plants (Figure 9). The accumulation of H2O2 and superoxide anion (O2−) was detected using 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) staining. The leaves of WT plants displayed darker spots than the transgenic Arabidopsis lines under salinity and drought stress conditions, which clearly indicates high accumulation of H2O2 and O2−in WT plants. The transgenic lines exhibited slightly lower accumulation of H2O2 under drought and salinity as indicated by the DAB staining. Additionally, the NBT staining clearly indicates a lower accumulation of O2- in transgenic plants, compared to the WT plants. This indicates that the transgenic plants had a lower accumulation of ROS, compared to the WT plants (Figure 9). 11 of 21 11 of 21 11 of 21 Int. J. Mol. Sci. 2019, 20, 2871 Int. J. Mol. Sci. 2018, 19, x FOR Int. J. Mol. Sci. 2018, 19, x FOR Figure 9. The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining Figure 9. The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining of WT and transgenic Arabidopsis plant leaves under salinity and drought stress conditions. Figure 9. The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining of WT and transgenic Arabidopsis plant leaves under salinity and drought stress conditions. Figure 9. The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining Figure 9. The 3,3-diaminobenzidine (DAB) and nitro blue tetrazolium (NBT) histochemical staining of WT and transgenic Arabidopsis plant leaves under salinity and drought stress conditions. Figure 9. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc However, the high-affinity K+ transporter 1 (HKT1) and SOD genes were upregulated in transgenic lines compared to WT plants, The results showed that the expression of a putative cation/H+ exchanger (CHX20) and a vacuolar Na+/H+ antiporter gene was downregulated in transgenic lines, compared to the WT plants under drought and salinity. Similarly, the expression of salt overly sensitive 1 gene (SOS1) and ABA stress-induced gene (ABA) was downregulated under drought stress. However, the high-aﬃnity K+ transporter 1 (HKT1) and SOD genes were upregulated in transgenic lines compared to WT plants, under salinity stress (Figure 10). are known to be involved in salinity stress tolerance mechanisms in plants. The results showed that the expression of a putative cation/H+ exchanger (CHX20) and a vacuolar Na+/H+ antiporter gene was downregulated in transgenic lines, compared to the WT plants under drought and salinity. Similarly, the expression of salt overly sensitive 1 gene (SOS1) and ABA stress-induced gene (ABA) was downregulated under drought stress. However, the high-affinity K+ transporter 1 (HKT1) and SOD genes were upregulated in transgenic lines compared to WT plants, under salinity stress (Figure 10). nder salinity stress (Figure 10). Figure 10. Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na+/H+ antiporter, ABA stress- induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a significant difference f h l ( 0 05) Figure 10. Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na+/H+ antiporter, ABA stress- induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a significant difference from the control (p < 0.05). Figure 10. Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na+/H+ antiporter, ABA stress-induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from the control (p < 0.05). Figure 10. Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na+/H+ antiporter, ABA stress- induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a significant difference Figure 10. 3. Discussion Date palm is a relatively stress-tolerant plant, but it has been lately observed that drought and salinity aﬀect the health of the plant to a remarkable extent, which reduces the productivity. To obtain molecular insights into the salinity tolerance mechanisms of date palm, various approaches, including transcriptome, methylome, metabolome and microbiome global analyses, have recently been employed [7,11,12,14,40,41]. Functional yeast analysis of the cDNA library was undertaken, which resulted in the identiﬁcation of several genes (including the date palm PdMT2A gene) that could play a critical role in salinity tolerance in date palm [18]. MTs are well known for detoxiﬁcation of heavy metal homeostasis [28,42,43]. However, MTs have also been shown to play a role in drought and salinity tolerance [33,44]. In this study, an attempt was made to functionally characterize the importance of the PdMT2A gene in salt, drought and oxidative stress tolerance. g g The deduced amino acid sequence of the PdMT2A gene shares common features with other metallothioneins, previously characterized from other plant species. These features include the presence of speciﬁc cysteine residues and the hydrophobicity proﬁle. In addition, the phylogenetic analysis showed a high similarity between the PdMT2A gene and its orthologs in other plant species, and therefore members of this gene family in the plants share a common ancestor gene (Figure 1A). These results suggest that MTs form part of the basic stress tolerance mechanisms in plants [29,33]. In fact, this evolutionary relationship amongst the plant MTs was demonstrated earlier in the genome-wide study of MT-family genes from Arabidopsis thaliana and various Brassica species [28]. Consistently, the putative promoter region of PdMT2A has been found to possess a signiﬁcant percentage of abiotic-stress-responsive TFBSs [45]. Amongst the TFBSs, MYB, bZIP, AP2/ERF and WRKY are known to be involved in abiotic stress responses in plants [46–50] (Figure 1D). Previously, transcriptome analysis of the leaf and root tissues of date palm showed that various genes of MYB, AP2/ERF, WRKY, NAC and bHLH transcription-factor families were co-overexpressed in response to salinity, and the abundance of these TFBSs was higher in root tissues than in the leaf tissues [12]. Interestingly, the TaMYB2 gene of wheat is used as a molecular marker for drought stress tolerance in common wheat [51]. The characterization of the HaAP2/ERF transcription-factor family showed that the AP2/ERF-family genes were overexpressed as a result of drought, cold, salt and heat treatments [52]. 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Tolera 2.4. Arabidopsis Seedlings Overexpressing PdMT2A Exhibit Improved Drought and Salinity Toleranc Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na+/H+ antiporter, ABA stress- induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a significant difference from the control (p < 0.05). Figure 10. Gene expression analysis of CHX20, SOS1, HKT1, vacuolar Na+/H+ antiporter, ABA stress-induced gene and SOD in WT and transgenic Arabidopsis subjected to salinity and drought stress. The bars represent the relative fold change (± SE, n = 3), while the asterisks indicate a signiﬁcant diﬀerence from the control (p < 0.05). Int. J. Mol. Sci. 2019, 20, 2871 12 of 21 12 of 21 3. Discussion These observations were consistent with previous reports in which overexpression of the GhMT3a gene from cotton led to an enhanced tolerance of H2O2 toxicity [34], and the overexpression of the SsMT2 gene from Suaeda salsa L. led to an enhanced tolerance of salinity and oxidative stress in yeast [24]. In addition, in transgenic E. coli cells, the OsMT3a gene of rice confers salinity and heavy metal stress tolerance [29]. The transgenic Arabidopsis seedlings had longer roots and slightly larger leaves, coupled with a higher biomass accumulation, compared to WT plants under drought, salinity and oxidative stress conditions (Figure 4). These observations support the notion that PdMT2A plays a role in abiotic stress tolerance, as previously reported for other plant MTs [24,33]. p y p p Plants maintain a stable regulation of Na+ and K+ homeostasis in the cytosol in the normal functioning of cellular and physiological processes [59]. The Na+ accumulation in PdMT2A transgenic and WT plants was similar under control conditions; however, under salinity the transgenic plants had a signiﬁcantly (p < 0.05) lower Na+ concentration and a higher K+/Na+ ratio than WT plants. These results are similar to those of an earlier report in which SsMT2 overexpression was shown to have similar eﬀects [24]. Plants have various strategies to modulate the inﬂux and eﬄux of Na+ and K+, including ion transporters such as NHX, CHX, HKT and Na+/H+ antiporters [41]. The plant MTs including PdMT2A are not previously known to play a role in Na+ and K+ transportation, however diﬀerential accumulation of Na+ and K+ ions was observed as a result of the overexpression of PdMT2A. Therefore, we ask what could contribute to the low Na+ accumulation in transgenic lines, compared to WT plants grown under salt stress. We speculate that the abundant PdMT2A could indirectly decrease the Na+ uptake, increase the activity of transporters that pump Na+ out from the plant cells or aﬀect both processes. Indeed, gene expression analysis revealed the regulatory eﬀect of PdMT2A on some ion transporter genes (Figure 10) under stress. This eﬀect may modulate the accumulation of Na+ and K+ ions in transgenic plants. For example, the downregulation of CHX20 and upregulation of SOS1 in transgenic plants under salinity could be a reason for the reduction of Na+ ions concentration. However, upregulation of HKT1 under salinity in the transgenic plants might have contributed to the accumulation of K+ ions. 3. Discussion Additionally, the watermelon ClWRKYs had a diverse response to abiotic stresses, which suggests that WRKYs positively or negatively participate in plant tolerance of drought, salt or cold stress [53]. These observations argue in favor of the potential involvement of PdMT2A in abiotic stresses and are consistent with previous observations on plant MTs [34,54,55]. p p The recombinant PdMT2A protein produced in E. coli had high initial velocity of the reaction when DTNB was used as a substrate. The MTs are one of the main sources of thiol in cells, and members of the thiol group are preferred targets for free radicals, which can be compared to the reduction of the disulﬁde bond of DTNB [56]. In addition, the cysteine residues in the proteins, like MTs, are highly reactive towards oxidizing agents including ROS [57]. Hence, the high initial speed of the reaction might be correlated with the high antioxidant capability of the PdMT2A protein (Figure 2C). However, a detailed study regarding the chemistry and protein folding of PdMT2A is required. When PdMT2A was overexpressed in yeast, it did not enhance the growth on solid media supplemented with NaCl, suggesting that PdMT2A is not part of the salt tolerance mechanism in yeast (Figure 3B). A similar pattern for the growth rate of yeast cells was observed previously when the rgMT gene of rice was overexpressed in yeast and assessed for salt tolerance [58]. Unlike in solid media, when the yeast cells were grown in liquid media (LSM) supplemented with NaCl, the TY cells showed slightly enhanced growth rates in the lag and the exponential growth phase compared to EV cells (Figure 3C). This enhanced growth rate could possibly be due to elevated early detoxiﬁcation of the ROS in the TY cells in the liquid medium [34]. Apparently, there was no signiﬁcant diﬀerence in the accumulation of Na+ and K+ by yeast cells under salinity stress. These results suggest that PdMT2A might not play a signiﬁcant role in maintaining the Na+ and K+ balance in yeast cells (Figure 3D). Int. J. Mol. Sci. 2019, 20, 2871 13 of 21 This ﬁnding is consistent with a previous study done on yeast cells transformed with the SsMT2 gene of Suaeda salsa L. [24]. The TY yeast cells displayed enhanced growth rates under oxidative and drought stresses (Figure 3B), suggesting that the transgenic yeast overexpressing PdMT2A possesses enhanced antioxidant activity. 3. Discussion Hence, this diﬀerential gene expression might be responsible for the balanced K+/Na+ ratio found in the transgenic plants under salinity. This notion may suggest a role for the PdMT2A in gene expression regulation probably through the activation of transcription factors such as the previously reported zinc ﬁnger transcription factor, which was regulated by metallothionein–thionein conjugate pair [39]. However, further studies are needed to determine the exact reason for lower Na+ accumulation in the transgenic lines overexpressing PdMT2A. The higher total chlorophyll content of the transgenic plants compared with the WT plants under abiotic stresses, especially under salinity stress, implies that the integrity of the photosynthesis system was maintained in the transgenic lines, due to the PdMT2A transgene (Figure 7A). A previous study also reported an increase in chlorophyll content under drought, salinity and heavy metal stress, in transgenic tobacco overexpressing the SbMT2 gene [60]. In addition, the TaMT3 gene of Tamarix androssowii led to high chlorophyll content in transgenic tobacco under heavy metal stress [43]. Proline is an osmoprotectant that maintains turgor pressure inside the plant cells during dehydration and high salinity stresses and also protects plants from oxidative damage. Hence, an increase in proline content under stress is considered to be an important phenomenon [61,62]. In this study, the proline concentration in the transgenic Arabidopsis lines was signiﬁcantly (p < .05) high under drought and salinity (Figure 7B). Intriguingly, proline concentration was signiﬁcantly (p < 0.05) lower in the transgenic lines grown under control conditions. Previous studies have reported the antioxidant ROS-scavenging activity of proline [63]. Therefore, the presence of a heterologous ROS scavenger such as PdMT2A in transgenic plants may have led to the reduction of proline under Int. J. Mol. Sci. 2019, 20, 2871 14 of 21 non-stress conditions. The histochemical staining for ROS detection also showed a reduction in ROS accumulation in transgenic plants under drought and salinity (Figure 9). This may imply an important role of PdMT2A in reducing the ROS routinely produced in the plants due to various biochemical reactions. This may also aﬀect the energy conservation strategy of plants by manipulating their metabolism. Under stress conditions, the fact that plants tend to accumulate ROS and maintain ROS homeostasis via scavenging enzymes such as SOD and APX, is well documented [64]. The overexpression of PdMT2A in Arabidopsis increased the SOD activity of transgenic plants compared to WT plants, under drought, salinity and oxidative stresses. 3. Discussion This was consistent with previous reports in which high SOD expression was observed under NaCl or osmotic stress conditions in transgenic tobacco overexpressing the SbMT2 gene [60]. The greater SOD levels probably reﬂect greater ROS-scavenging activity, imparting improved abiotic stress tolerance (Figure 7D). The APX activity in transgenic Arabidopsis did not vary compared with WT plants, under salinity and oxidative stress conditions. However, there was an increasing trend of the APX activity in transgenic plants subjected to drought stress (Figure 7E), and this trend was also observed in transgenic tobacco expressing SbMT2 [60]. 4.1. Sequence Analysis of Date Palm Metallothionein PdMT2A homologous sequences from other plant species were retrieved from the NCBI database (https://www.ncbi.nlm.nih.gov/) for the sequence comparison. The sequences were aligned using ClustalW and a phylogenetic tree was constructed using the neighbor joining method implemented via the MEGA X software package [65]. The physicochemical properties of the PdMT2A protein were analyzed using the ProtParam tool (https://web.expasy.org/protparam/) and the mean hydrophobicity index was plotted according to the Kyte–Doolittle scale [66,67]. The putative 2000-bp upstream promoter sequence was analyzed for cis-binding sites using PlantPAN 2.0 (http://PlantPAN2.itps. ncku.edu.tw/) [68]. The results were analyzed, and a pie diagram representing the distribution of transcription factor binding sites (TFBSs) was produced. 4.2. Recombinant Protein Production and Reaction with DTNB The PdMT2A transcript was ampliﬁed via PCR from the date palm cDNA using forward (5′-GGATTTCCATATGATGTCTTGCTGTAGCGG-3′) and reverse (5′-GGATTTCCATATGATGTCTTG CTGTAGCGG-3′) primers. The amplicon was designed to include the Nde1 and EcoR1 restriction sites, in order to be compatible with the same sites in the pTYB21 plasmid (New England Biolabs, Ipswich, MA, USA). The resulting plasmid construct was named pTYB21-PdMT2A. After ampliﬁcation in the Escherichia coli DH10B strain, the construct was transformed into the E. coli ER2566 strain using the standard electroporation method, to produce the recombinant protein. A selected transformed E. coli colony was inoculated into 0.5 L of Luria-Bertani (LB) medium for 16 h at 37 ◦C with agitation at 200 rpm. Recombinant protein production was induced by adding 250 µL of 1 M isopropyl β-d-1-thiogalactopyranoside (IPTG) and incubating at room temperature for 16 h with agitation at 200 rpm. Subsequently, the recombinant protein puriﬁcation was carried out via in-column aﬃnity chromatography with chitin beads, using an Impact™Kit and following the manufacturer’s instructions (New England Biolabs). In addition, 1 mM ethylenediaminetetraacetic acid (EDTA) was included in the protein extraction buﬀer in order to reduce the metal binding prior to the reaction with the substrate. The intein tag contains a chitin-binding domain (CBD) for aﬃnity puriﬁcation of the fusion protein on a chitin resin and was cleaved using dithiothreitol (DTT), which also prevents the formation of disulﬁde bridges between groups of cysteine residues of the PdMT2A. The amount of protein recovered was quantiﬁed using the standard curve of glutathione (GSH), as described earlier [69]. The reactivities of the recombinant PdMT2A protein and the corresponding empty vector control protein, were measured in a 100-µL reaction vessel containing 15 of 21 Int. J. Mol. Sci. 2019, 20, 2871 0.5 mM 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) as a substrate, 0.2 M phosphate buﬀer (pH 8.0) and 0.5 mM of the recombinant protein [70]. The formation of 2-nitro-5-thiobenzoic acid (TNB) was detected by monitoring the absorbance at 412 nm for 60 min. The initial speed of the reaction was obtained by plotting a curve of absorbance against time, using a spectrophotometer, as previously described [71]. 4.3. Expression of PdMT2A in Salt-Sensitive Yeast Strain The full-length PdMT2A gene was obtained from the date palm cDNA library and cloned into the yeast expression vector pYES-DEST52 (Thermo Fisher Scientiﬁc, Carlsbad, CA, USA), using site-speciﬁc Gateway™recombination technology (Thermo Fisher Scientiﬁc) downstream of the galactose-inducible GAL1 promoter (Figure 3A). The recombinant pYES-DEST52-PdMT2A vector (TY) and the empty pYES-DEST52 vector (EV) were transferred to the salt-sensitive mutant yeast (S. cerevisiae) strain BYT458 [72] (kindly provided by Hana Sychrova, Czech Republic), using Yeastmaker™Yeast Transformation System 2 (Clontech Laboratories, Inc., Mountainview, CA, USA), following the manufacturer’s instructions. The yeast cells were grown in liquid synthetic medium (LSM) supplemented with 2% glucose. Subsequently, the yeast cells were spotted on solid synthetic medium (SSM) supplemented with 2% galactose, with 2% PEG (polyethylene glycol), 300 mM NaCl (sodium chloride), 10 mM LiCl (lithium chloride) or 3 mM H2O2 (hydrogen peroxide), as drought, salinity, Li toxicity and oxidative stresses, respectively. Ten-µL drops of serially diluted yeasts were spotted on the SSM plates and the plates were incubated at 30 ◦C for ﬁve days before the observations were made [18]. The growth rates of TY and EV cells were monitored by growing the cells in 20 mL of LSM supplemented with 2% galactose and 50 mM NaCl and incubated in a shaker-incubator at 30 ◦C with agitation at 200 rpm. The optical density (OD) of the liquid was measured every 12 h for three days. Similarly, to monitor the uptake and accumulation of Na+ and K+ in yeast, the TY and EV cells were grown in LSM supplemented with 25 mM NaCl, and Na+ and K+ concentrations were measured using a ﬂame photometer, as described previously [73]. 4.4. Generation of Transgenic Arabidopsis Plant Overexpressing PdMT2A Gene A full-length PdMT2A gene was cloned in the binary plant expression vector pEarleyGate 203 (TAIR stock ID—CD3-689) in a frame with an Myc-tag protein and overexpressed under the control of the 35S CaMV constitutive in-plant promoter. The pEarleyGate 203-PdMT2A construct was ampliﬁed in the E. coli DH10B strain and further transformed into Agrobacterium tumefaciens LBA4404 strain by electroporation. To produce transgenic Arabidopsis lines, 45-day-old wild-type (WT) Arabidopsis thaliana Columbia (Col-0) plants were transformed using the standard Agrobacterium-mediated ﬂoral dip method [74]. The T0 seeds were harvested from the plants, vernalized at 4 ◦C and sown on soil. The transgenic plants were selected by spraying T1 plants with 0.01% Basta® (Bayer, Germany) herbicide solution on the 6th and 10th day after gemination. The surviving transgenic plants (T1) were conﬁrmed by PCR using 35S promoter forward (5′-CAAGACCCTTCCTCTATATAAG-3′) and OSC terminator reverse (5′-CGCATATCTCATTAAAGCAG-3′) primers. The T2 seeds were harvested, dried, vernalized and germinated on half-strength Murashige and Skoog (MS) medium supplemented with 10 mg/L of Basta® herbicide. The transgenic T2 lines, showing the 3:1 Mendelian segregation ratio for resistant:sensitive plants, were selected and planted in soil, and the T3 seeds were collected from them. Three independent homozygous transgenic lines were obtained by screening T3 seeds for 100% herbicide resistance and used for further experiments. 4.7. Determination of Na+, K+, Chlorophyll, Proline and Malondialdehyde Content and Antioxidant Enzyme Activities 4.7. Determination of Na+, K+, Chlorophyll, Proline and Malondialdehyde Content and Antioxidant Enzyme Activities Arabidopsis seedlings were collected from the control, drought and salt-treated MS plates and dried, weighed and digested in 10 mL of a 0.1 M nitric acid solution before incubation in a shaker (100 rpm) for two days at room temperature. The mixture was ﬁltered using Whatman No. 1 ﬁlter paper and the Na+ and K+ concentrations were measured using a ﬂame photometer, as previously described [75]. The total chlorophyll content was measured using the 80% acetone-based method [76]. Proline concentration was determined by homogenizing plant tissue in 3% aqueous sulfosalicylic acid and allowing further reaction with acid ninhydrin and glacial acetic acid at 100 ◦C for one hour. The reaction mixture was cooled in ice and extracted by adding 1 mL of toluene followed by vortex mixing for 20 s. The absorbance was measured at 520 nm and the proline concentration was determined from a standard curve, as described previously [77]. Malondialdehyde (MDA), a product of lipid peroxidation, was determined spectrophotometrically, as described earlier [78]. Brieﬂy, plant tissue was homogenized in 1 mL of 20% trichloroacetic acid containing 0.5% thiobarbituric acid in solution, and the mixture was heated to 95 ◦C for 30 min and centrifuged at 10,000 rpm for 10 min. The absorbance was measured at 532 nm and the value of the non-speciﬁc absorbance at 600 nm was subtracted. 1 1 p The concentration of MDA was calculated using its extinction coeﬃcient of 155 mM−1·cm− The crude enzyme extract was isolated from transgenic and WT Arabidopsis seedlings using an extraction buﬀer containing 50 mM potassium phosphate buﬀer (KPB) (pH 7.5), 1% polyvinylpolypyrrolidone (PVPP) and 2 mM EDTA. Superoxide dismutase (SOD) activity was determined based on inhibition of the photochemical reduction of nitro blue tetrazolium (NBT) with a reaction mixture containing 50 mM KPB, 75 mM NBT, 15 mM methionine, 2 mM riboﬂavin and 10 µL enzyme extract, when exposed to a 15-W ﬂuorescent lamp for 10 min. The absorbance was recorded at 560 nm and the SOD activity was calculated as described earlier [79]. The ascorbate peroxidase (APX) enzymatic activity was determined by estimating the reduction of H2O2 by APX at 290 nm. The reaction mixture was composed of 50 mM KPB, 0.5 mM ascorbate, 0.1 mM H2O2 and 10 µL enzyme extract. 4.5. Detection of Overexpressed PdMT2A Protein in Arabidopsis Using Dot-Blot Immunoassay The leaves of the transgenic Arabidopsis were harvested, ﬂash-frozen and crushed to ﬁne powder using liquid nitrogen. The total protein was extracted using 1 mL of extraction buﬀer composed of 100 mM tris-HCl (pH 7.5), 100 mM NaCl, 1 mM phenylmethyl sulfonyl ﬂuoride (PMSF) and 5% glycerol. Since the PdMT2A is too small in size (approximately 8 kDa) and is unstable in air, it was not Int. J. Mol. Sci. 2019, 20, 2871 16 of 21 possible to detect this protein using the traditional Western blot assay. Therefore, the overexpressed PdMT2A protein fused to the Myc tag was detected via the dot-blot method using 0.2 µM PVDF membrane (Bio-Rad, Hercules, CA, USA), the anti-Myc-tag primary polyclonal antibody ab117499 (Abcam, Cambridge, UK) (dilution 1:1000) and the anti-mouse IgG (H&L) horseradish peroxidase (HRP) conjugated secondary antibody ab205719 (Abcam) (dilution 1:1000). As a protein loading control in the dot-blot assay, an immunoassay was carried out using the polyclonal anti-rabbit primary antibody AS13 2640 (Agrisera, Vännäs, Sweden) against the actin protein and the HRP-linked secondary anti-rabbit antibody AS09 602 (Agrisera). Immunoreactions were visualized by detecting the chemiluminescence signal on the immunoblot using Clarity enhanced chemiluminescence (ECL) substrate (Bio-Rad) and the image was visualized using the ChemiDoc™Touch Imaging System (Bio-Rad). 4.6. Stress Treatment Assays of the Transgenic Arabidopsis Plants The ability of the transgenic Arabidopsis lines to tolerate stress was compared with the wild-type (WT) Arabidopsis plants, both on plates containing MS medium (seedlings) and on soil (adult plants), in growth chambers maintained at 22 ◦C and 70% relative humidity (RH) with a 16-h-day/8-h-night cycle. For seedling assays, the seeds were initially germinated on plates with half-strength MS medium for four days, and later transferred to plates containing MS medium or plates supplemented with 100 mM NaCl, 150 mM mannitol or 2 mM H2O2, representing salinity, drought and oxidative stresses, respectively. Seedlings were grown for 14 days and then data (root lengths and dry weights) were collected. Similarly, for evaluating the stress tolerance of plants grown on soil, 21-day-old plants were either irrigated with 200 mM NaCl solution (salt stress) or watering was withheld for 14 days (drought stress). 4.10. Statistical Analysis One-way analysis of variance (ANOVA) was used, with Tukey’s post hoc test, to determine the statistically signiﬁcant diﬀerences (p < 0.05) between the means of tested parameters. 4.9. Expression Analysis of Abiotic-Stress-Responsive Genes using qPCR 4.9. Expression Analysis of Abiotic-Stress-Responsive Genes using qPCR RNA was extracted from the WT and transgenic Arabidopsis plants using a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions. A total of 100 ng of RNA was converted into cDNA using a SuperScript™IV First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. The quantitative real-time PCR (qPCR) gene expression analysis was performed for six stress-responsive genes, using gene-speciﬁc primer pairs (Table S1). A 20-fold diluted cDNA was used with SsoAdvanced™Universal SYBR® Green Supermix (Bio-Rad) for qPCR, in a CFX96 Touch™Real-Time PCR Detection System (Bio-Rad). The Arabidopsis actin (AtActin accession number AT3G18780) was used as a reference gene to normalize the expression data, using the 2−∆∆Ct method [82]. 4.7. Determination of Na+, K+, Chlorophyll, Proline and Malondialdehyde Content and Antioxidant Enzyme Activities The APX activity was determined using an extinction coeﬃcient of 2.8 mM−1·cm−1 [80]. Int. J. Mol. Sci. 2019, 20, 2871 17 of 21 17 of 21 4.8. Detection of Superoxide Anion and H2O2 Accumulation in the Leaves of Arabidopsis 4.8. Detection of Superoxide Anion and H2O2 Accumulation in the Leaves of Arabidopsis H2O2 accumulation was detected in leaves by staining with 3,3-diaminobenzidine (DAB), and superoxide anion (O2¯) was detected with nitro blue tetrazolium (NBT). Brieﬂy, the stressed and non-stressed Arabidopsis leaves were immersed in 0.1% DAB solution and 0.2% NBT solution and incubated at room temperature overnight. The leaves were decolorized by immersing them in absolute ethanol and boiling for 15 min [81]. 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PLoS ONE 2016, 11, e0166216. [CrossRef] [PubMed] 14. 5. Conclusions The study showed that the PdMT2A gene improved drought and salinity tolerance in yeast. In addition, the transgenic Arabidopsis plants performed better than the WT plants under drought, salinity and oxidative stress conditions. The transgenic plants had maintained a high K+/Na+ ratio which could be partly attributed to an indirect eﬀect of the PdMT2A transgene on regulating the plant ion transporters. The transgenic plants maintained high chlorophyll retention under stress conditions, indicating the integrity of the photosynthesis system. Additionally, transgenic plants had high proline content and relatively lower ROS levels under drought and salinity stress conditions. Collectively, PdMT2A overexpression improved stress tolerance in transgenic Arabidopsis plants by maintaining chlorophyll, high K+/Na+ ratio, high proline content and decreased ROS levels under drought and salinity stresses. Overall, this study represents one of the very few reports that functionally characterized stress-responsive genes from the date palm. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/12/ 2871/s1. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/12/ 2871/s1 Author Contributions: H.V.P. conceived, designed, performed the experiments, analyzed data, and wrote the manuscript; I.A.-H. performed the experiments and revised the manuscript, R.A.-Y. revised the manuscript; L.A.K. contributed reagents/materials/analysis tools, G.A.J. performed the experiments, R.S. revised, edited and provided suggestions on the manuscript, and M.W.Y. designed the experiment, supervised the work, wrote the manuscript and contributed reagents/materials/analysis tools. Funding: This study is supported by the generous grant number 151 from the research council (TRC), Oman to M.W.Y. 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https://openalex.org/W2735394585	https://europepmc.org/articles/pmc5512835?pdf=render	English	null	Elevated serum CEA levels are associated with the explosive progression of lung adenocarcinoma harboring EGFR mutations	BMC cancer	2,017	cc-by	6,649	Gao et al. BMC Cancer (2017) 17:484 DOI 10.1186/s12885-017-3474-3 Gao et al. BMC Cancer (2017) 17:484 DOI 10.1186/s12885-017-3474-3 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. * Correspondence: spp128@126.com 2Department of Thoracic Surgery, Shandong Tumor Hospital and Institute, Jinan, Shandong Province 250117, China 4Department of Thoracic Surgery, Shandong Cancer Hospital and Institute, Jiyan Rd. 440, Jinan, Shandong 250117, People’s Republic of China Full list of author information is available at the end of the article Elevated serum CEA levels are associated with the explosive progression of lung adenocarcinoma harboring EGFR mutations Yuan Gao1, PingPing Song2,4, Hui Li2, Hui Jia3 and BaiJiang Zhang2 Abstract Background: Serum carcinoembryonic antigen (CEA) levels are a predictor of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) efficacy and are associated with epidermal growth factor receptor (EGFR) gene mutations. However, the clinical significance of plasma CEA level changes during different cycles of target therapy is unknown for lung adenocarcinoma patients with sensitizing EGFR mutations. Methods: In total, 155 patients with lung adenocarcinoma were enrolled in this retrospective study between 2011 and 2015. EGFR mutations were detected by RT-PCR (real-time quantitative PCR). Plasma CEA levels were measured prior to different EGFR-TKI treatment cycles. Computed tomography (CT) scans were conducted every 2 months to assess the therapeutic efficacy. Results: Serum CEA concentrations were significantly associated with EGFR mutations (p < 0.05). Furthermore, in all patients treated with EGFR-TKIs, the serum CEA levels increased with disease progression (p < 0.005). A COX multivariate analysis revealed that CEA levels 16.2 times above normal were associated with early disease progression (HR, 5.77; 95% CI:2.36 ~ 14.11; p < 0.001). Based on this finding, a threshold was set at the median time of 8.3 months. Patients with EGFR mutations exhibited a median progression-free survival time of 12.8 months. Serum CEA levels were markedly increased compared to levels measured 4.5 months prior to the changes detected via CT scans for patients resistant to EGFR-TKIs. Conclusions: Elevated CEA levels during targeted therapy may be a more sensitive predictor of explosive lung adenocarcinoma progression in patients harboring mutant EGFRs compared to traditional imaging methods. Keywords: Biomarker, EGFR, EGFR-TKIs, Mutation, Response mutations [2–7]. One such EGFR-TKI is the orally ad- ministered, targeted agent erlotinib, which inhibits the tyrosine kinase domain of the EGFR. Erlotinib is ap- proved for second-line use based on the positive results of a phase 3 BR.21 trial [8] in which erlotinib improved overall survival (OS) compared with the best supportive care. Erlotinib also has clinical benefits as a first-line therapy for advanced NSCLC. The tumor response rate was 10-20%, and the median survival duration was 10.9- 12.9 months in phase 2 studies [9, 10]. However, almost all patients suffered from tumor progression and inevit- ably became resistant to EGFR-TKIs within 8-12 months (a phenomenon referred to as acquired resistance). Background g Lung cancer is the leading cause of cancer-related mor- tality worldwide, and non-small cell lung cancer (NSCLC) is the most common form of lung cancer. Many NSCLC patients present with an advanced disease stage upon initial diagnosis [1]. Patients with tumors that harbor activating mutations in the epidermal growth factor receptor (EGFR) benefit greatly from treatment with EGFR tyrosine kinase inhibitors (TKIs) compared with patients whose tumors lack these Correspondence: spp128@126.com 2Department of Thoracic Surgery, Shandong Tumor Hospital and Institute, Jinan, Shandong Province 250117, China 4Department of Thoracic Surgery, Shandong Cancer Hospital and Institute, Jiyan Rd. 440, Jinan, Shandong 250117, People’s Republic of China Full list of author information is available at the end of the article Currently, the standard method of measuring the effi- cacy of a lung cancer treatment is anatomical imaging, Gao et al. BMC Cancer (2017) 17:484 Gao et al. BMC Cancer (2017) 17:484 Page 2 of 7 Page 2 of 7 simultaneously. All responses were defined according to the Response Evaluation Criteria in Solid Tumors (RECIST) criteria. A response was confirmed at 4 weeks (for a complete or partial response) or 6 weeks (for stable disease) after the first documentation. Other in- clusion criteria included an Eastern Cooperation Oncol- ogy Group (ECOG) performance status of 0-3 [13]. Patients who had received prior treatment with oral EGFR-TKIs or were allergic and/or intolerant to these drugs were excluded from the study. The progression- free survival (PFS) and overall survival (OS) of the meta- bolic responders and non-responders were the end points of the study. The baseline patient characteristics are presented in Table 1. This study complied with the guidelines of the local ethics committee. including computed tomography (CT) scanning, which measures the size of malignant lesions before and after treatment. However, the use of anatomical imaging methods to assess treatment efficacy presents a number of drawbacks, the most critical of which is a delay in treatment due to changes in tumor size. Furthermore, the efficacy of targeted drugs is often not reflected by a change in tumor size but rather by changes in cell me- tabolism. Therefore, the identification of clinical bio- markers in patients with EGFR mutations may prove useful when anatomical analyses are not feasible. Several serum markers are considered to be prognostic and predictive markers of NSCLC. Determination of EGFR mutation Determination of EGFR mutation EGFR mutation analysis was performed via a fragment analysis using polymerase chain reaction (PCR) and the Cycleave real-time quantitative PCR techniques (SRL Inc., Tokyo, Japan). Background Among these markers, carcinoembryonic antigen (CEA) is a sensitive and useful tumor marker for cancer diagnosis and prognosis and the assessment of therapy [6–8]. According to recent reports, CEA is a significant predictor of sensitivity and survival in patients treated with gefitinib [9–11]. The present study (1) compared the significance of CEA levels with other clinical characteristics (i.e., age, sex, smoking history, per- formance status [PS], and CYFRA1-1) and (2) determined whether the serum CEA levels correlated with EGFR-TKI resistance. This correlation would permit the use of CEA as a biomarker in NSCLC patients and would aid in iden- tifying treatment candidates with reversible and irrevers- ible EGFR-TKI resistance and candidates for whom an early intervention with combined chemotherapy and radiotherapy is more appropriate. Measurement of serum tumor marker levels Serum CEA (normal range: 0-3.4 ng/ml) and CYFRA 21-1 (normal range: 0-3.3 ng/ml) were measured via an electrochemiluminescence immunoassay on an auto- matic analyzer (Elecsys200; Roche Diagnostics Mann- heim, Basel, Switzerland) before TKI treatment. Patient characteristics Discussion EGFR mutation predicts the efficacy of EGFR-TKIs in patients with advanced NSCLC. However, acquiring an adequate tissue sample for an EGFR mutational analysis is not often feasible, particularly in patients with ad- vanced disease [2, 8, 14]. A recent study reported that Patient characteristics The clinicopathological characteristics of the 155 pa- tients are summarized in Table 1. Ninety-two patients (65.8%) were women, and 92 patients (65.8%) were non- smokers. The patient age ranged from 38 to 81 years (median: 60 years). Fifty-seven patients were classified as pathological stage classes I and II, and 98 patients were classified as III and IV. One hundred fifty-two patients (98.1%) had a PS of 0-1, and three (1.9%) patients had a PS of 2-3. The median OS and PFS were 28.5 and 12.8 months, respectively. A single factor χ2 test showed that EGFR mutation was associated with gender, age, smoking history, and the serological levels of CEA and CYFRA 21-1 times higher than normal) in elevated group. According to the Cox regression analysis, CEA levels 16.2 times higher than normal at any point during the 2 months prior to the determination of progress represented an early response to disease progression (HR, 5.77; 95% CI:2.36 ~ 14.11; p < 0.001; Table 4). We noted that the median time until this threshold (i.e., CEA levels at least 16.2 times higher than normal) was reached was 8.3 months. However, for patients with EGFR mutations, the median was 12.8 months. Methods In total, 155 primary lung adenocarcinoma patients, who had been hospitalized at the Department of Respiratory, Oncology and Thoracic Surgery in Shandong Provincial Tumor Hospital between August 8, 2011, and March 8, 2015, were enrolled in this retrospective study. The en- rolled patients tested positive for an EGFR mutation and had received EGFR-TKI as a 1st, 2nd, or 3rd line of treatment [6, 12]. Patients with locally advanced (stage IIIB), metastasized (stage IV), or post-surgically relapsed NSCLC were confirmed for EGFR mutations and re- ceived either gefitinib 250 mg/d or erlotinib 150 mg/d orally. The clinical stage was determined by the tumor, node, and metastasis (TNM) classification system (7th edition). The following inclusion criteria were utilized for this study: patients had pathologically identified adenocarcinoma; had received an initial therapy (includ- ing chemotherapy, surgery or chemoradiotherapy); and had survived for more than 1 month. Histological sub- classification was performed according to the World Health Organization classification. A CT scan was per- formed to assess the tumor size within 28 days prior to initiating treatment and was repeated every 2 months. Serum tumor markers and CT scans were measured Table 1 Patient characteristics Characteristic No. of Patients Percentage Sex Male 63 34.2% Female 92 65.8% Clinical Stage I,II 57 36.8% III,IV 98 63.2% Smoking history Ever 63 34.2% Never 92 65.8% Age,y < 59 74 47.7% ≧59 81 52.3% ECOG PS 0-1 152 98.1% 2-3 3 1.9% Median age of patients 59y (range,38-81y) Patient characteristics Gao et al. BMC Cancer (2017) 17:484 Gao et al. BMC Cancer (2017) 17:484 Page 3 of 7 Table 2 Analysis of EFGR mutation Clinical Characteristic Sample(n) EGFR mutation(n) χ2 P Gender Female 92 47 3.957 0.047 Male 63 22 Age < 59 74 30 4.230 0.040 ≧59 81 39 Stage I,II 57 21 2.150 0.143 III,IV 98 48 PS Score 0-1 152 68 0.155 0.694 2-3 3 1 Smoker Ever 63 22 3.957 0.047 Never 92 47 CEA < 3.4 ng/ml 63 21 5.374 0.020 ≧3.4 ng/ml 92 48 Cyfra21-1 < 3.3 ng/ml 64 44 25.920 0.001 ≧3.3 ng/ml 91 25 A single factor χ2 test showed that EGFR mutation was associated with gender, age, smoking history, and the serological levels of CEA and CYFRA 21-1 Statistical methods There were no missing data in our study. We used SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA) for data processing. The χ2 test, Fisher’s exact test and multivariate logistic regression analysis were used to analyze the associations between EGFR mutations and clinical factors. Survival was estimated using the Kaplan- Meier method. Overall survival was measured as the date of the first course of initial therapy to the date of death or the last follow-up examination. A log-rank test was performed to evaluate significant differences in the overall survival among the groups. P values <0.05 were considered significant. A multivariate analysis using the Cox proportional hazards model was used to establish the association between the clinical variables and survival. Relationship between CEA levels and EGFR gene mutations A single factor χ2 test showed that EGFR mutation was associated with gender, age, smoking history, and the serological levels of CEA and CYFRA 21-1 (p < 0.05; Table 2). The multivariate logistic analysis revealed that patient gender and serological CEA levels were corre- lated with EGFR mutation (p < 0.05) (Table 3). A ROC curve was drawn, and the area under the curve was calculated. The area under the curve for CEA was 0.567 (95% CI: 0.476 ~ 0.657, p < 0.001). When the CEA cut-off was 3.4 ng/ml, the sensitivity was 69.6% and the specificity was 48.8% (Figure 1). TKIs We evaluated the efficacy of EGFR-TKIs via CT scanning and serum tumor marker concentrations (CEA) every 2 months. The patients were divided according to serum CEA levels into elevated and non-elevated groups in progress group by CT scanning. A χ2 test revealed that increased serum CEA levels were related to disease pro- gression (p < 0.005). Patients were then divided into two groups according to the average value (CEA levels 16.2 Table 3 Futher analysis of the association of EGFR mutation Factor EGFR Mutation OR P 95%CI Gender 0.479 0.034 0.243 ~ 0.946 CEA 2.529 0.001 1.283 ~ 4.984 The multivariate logistic analysis revealed that patient gender and serological CEA levels were correlated with EGFR mutation Table 3 Futher analysis of the association of EGFR mutation The multivariate logistic analysis revealed that patient gender and serological CEA levels were correlated with EGFR mutation Gao et al. BMC Cancer (2017) 17:484 Page 4 of 7 Page 4 of 7 fragments carrying tumor specific sequence alterations cftDNA are found in the cell-free fraction of blood, representing a variable and generally small fraction of the total circulating DNA. cftDNA has a high degree of specificity to detect EGFR gene mutations in NSCLC. Fragments of circulating DNA were isolated in plasma many years ago [18]. In particular, patients with cancers present higher levels of circulating DNA comparing to healthy volunteers because of the presence of tumoral counterpart, which express the same molecular abnor- malities expressed by DNA of primitive mass [19]. The elevate cellular turn over and consequent cellular necro- sis and apoptosis cause a massive release of tumoral DNA into the bloodstream were it can be isolated and analyzed. Therefore, tumor size, localization and vascu- larity may influence cftDNA plasmatic levels. It is also possible that part of cftDNA comes from CTCs lysis [19]. The analysis of cftDNA, defined as liquid biopsy, could be repeated every time needed and without any discomfort for patients. Moreover, the mutational ana- lysis of cftDNA demonstrated a signicantly better sensi- tivity if compared with CTCs one, establishing cftDNA as the best circulating source for molecular analysis [20]. Information derived from liquid biopsy could be used in future for early cancer diagnosis, assessment of genetic determinants for targeted therapies, monitoring of tumor dynamics and early evaluation of tumor response, identification of resistance mechanisms [19]. According to the Cox regression analysis, CEA levels 16.2 times higher than normal at any point during the 2 months prior to the det represented an early response to disease progression TKIs cftDNA could be a relevant biomarker to molecular diagnosis and monitor treatment resistance, because of its sensitiv- ity and specificity, but it really needs reproducible and standardized methods, both for the extraction and for its analyses. Regarding the mutation analysis of cftDNA, a large number of technologies is now available to analyze mutations in cftDNA, including automatic sequencing, real-time polymerase chain reaction (PCR) platforms, mass spectrometry (MS) genotyping, ampli cation proto- cols with magnetic beads in oil emulsions [beads, emul- sion, ampli cation and magnetics (BEAMing)] and next- generation sequencing (NGS), digital PCR platforms [21–25]. The sensitivity range of the available techniques Fig. 1 A ROC curve was drawn, and the area under the curve was calculated. The area under the curve for CEA was 0.567 (95% CI: 0.476 ~ 0.657, p < 0.001). When the CEA cut-off was 3.4 ng/ml, the sensitivity was 69.6% and the specificity was 48.8% fragments carrying tumor specific sequence alterations cftDNA are found in the cell-free fraction of blood, representing a variable and generally small fraction of the total circulating DNA. cftDNA has a high degree of specificity to detect EGFR gene mutations in NSCLC. Fragments of circulating DNA were isolated in plasma many years ago [18]. In particular, patients with cancers present higher levels of circulating DNA comparing to healthy volunteers because of the presence of tumoral counterpart, which express the same molecular abnor- malities expressed by DNA of primitive mass [19]. The elevate cellular turn over and consequent cellular necro- sis and apoptosis cause a massive release of tumoral DNA into the bloodstream were it can be isolated and analyzed. Therefore, tumor size, localization and vascu- larity may influence cftDNA plasmatic levels. It is also possible that part of cftDNA comes from CTCs lysis [19]. The analysis of cftDNA, defined as liquid biopsy, could be repeated every time needed and without any discomfort for patients. Moreover, the mutational ana- lysis of cftDNA demonstrated a signicantly better sensi- tivity if compared with CTCs one, establishing cftDNA as the best circulating source for molecular analysis [20]. Information derived from liquid biopsy could be used in future for early cancer diagnosis, assessment of genetic determinants for targeted therapies, monitoring of tumor dynamics and early evaluation of tumor response, identification of resistance mechanisms [19]. According to the Cox regression analysis, CEA levels 16.2 times higher than normal at any point during the 2 months prior to the determination of progress represented an early response to disease progression ession analysis, CEA levels 16.2 times higher than normal at any point during the 2 months prior to the determination of progress onse to disease progression TKIs BMC Cancer (2017) 17:484 Page 5 of 7 Page 5 of 7 molecular mechanisms may lead to EGFR-TKI resist- ance, it is important to non-invasively detect tumors re- fractory to EGFR-TKI treatment and identify the mechanisms underlying this resistance. Thus, the ther- apy could be effectively tailored to each patient. Based on previous reports, the function of CEA has not been elucidated. However, as a cell surface adhesion protein, CEA may play a role in cell-cell adhesion [36]. Overex- pression of CEA is thought to play a role in tumorigen- esis [37]. Furthermore, CEA has a dominant effect in blocking differentiation, and CEA cooperates with Myc and Bcl-2 during cellular transformation [38]. Further- more, CEA can inhibit cell death induced by a loss of anchorage to the extracellular matrix (anoikis) [39]. If CEA is upregulated following activation of the EGFR pathway, its serum levels may trigger an EGFR mutation. Although these findings suggest that CEA may have anti-apoptotic effects in cancer cells, a direct relation- ship between high CEA levels and patient responses to EGFR-TKIs has not yet been established and requires additional research. varies from 15 to 0.01%, but one of the major gaps in this field is the lack of standardization of techniques, in order to understand how those techniques are cost- effective and reliable to the clinical needs. Therefore, simpler and more accessible predictors of EGFR mutations, such as surrogate markers, are neces- sary. CEA is the product of the CEACAM5 gene, which is expressed only in epithelial cells. CEA is found more abundantly on the apical surface of the gastrointestinal epithelium but is also found in other mucosal epithelia cells, such as in the lung [26]. Although CEA was often falsely elevated in smokers and in patients with restrict- ive or obstructive pulmonary diseases [27–29], abnor- mally elevated CEA levels were reported in 30-70% of patients with NSCLC. Abnormally elevated CEA levels are most frequently observed in patients with adenocar- cinoma and advanced stage carcinoma [30]. In addition, high serum CEA levels are associated with a poor prog- nosis in patients with NSCLC, regardless of treatment [30, 31]. According to Japanese scholars, patients with elevated serum CEA levels responded better to gefitinib. Furthermore, recurrent lung adenocarcinoma patients with high serological CEA levels have a higher EGFR mutation rate after surgery and higher serological CEA levels. TKIs These findings are attributed to a possible anti- apoptotic signal in the mutant EGFR pathway that could elevate the expression level of the CEA protein [32]. However, the specimens used for genetic testing were surgical specimens obtained prior to disease recurrence and may not represent all the biological characteristics of a recurrent tumor [33]. In our study, the serum CEA level in the EGFR gene mutation group was significantly higher than in the non-mutated group. Both the univari- ate and multivariate analyses indicated that the serum CEA levels correlated with EGFR mutations (higher serum CEA levels were associated with higher EGFR gene mutation rates). Our data are similar to the find- ings of Okamato et al. [34]. Shoji et al. [35] reported that the rate of EGFR gene mutation significantly increased as the serum CEA levels increased (for serum CEA levels <5, ≥5 (but <20), and ≥20, the rates of EGFR gene mutation were 35, 55 and 87.5%, respectively; p = 0.040). Several reports have described the relationship be- tween serological markers and the curative effect of EGFR-TKIs. However, these reports did not perform EGFR mutation testing or dynamic monitoring of CEA levels to predict EGFR-TKI resistance. Therefore, these reports cannot determine the most effective treatment for early intervention. Despite the high responsiveness of tumors bearing activating EGFR mutations, almost all We found that a persistently high level of CEA after treatment with a reversible EGFR-TKI can successfully identify patients with NSCLC cells that are resistant (perhaps because of the occurrence in the EGFR kinase domain of a T790 M secondary mutation that prevents EGFR-TKI binding and subsequent growth arrest). Fur- thermore, when the CEA level was 16.2 times greater than normal, the elevation was associated with distant metastasis (Table 2). According to Sequist et al. [39], molecular analyses of repeated lung biopsies from these patients are needed to identify different mechanisms of acquired resistance. A potential clinical application of our observations could be the development of a test for patient responsiveness to EGFR-TKI treatment using non-invasive serum tumor markers. The information provided by this test may facilitate the selection of pa- tients as candidates for therapy with reversible or irre- versible EGFR-TKIs and the development of therapeutic strategies for overcoming resistance in patients with re- fractory NSCLC. Tumors with high CEA expression may possess an increased capacity to develop distant metasta- ses (perhaps due to vascular-tumoral cell-cell adhesion processes). TKIs cftDNA could be a relevant biomarker to molecular diagnosis and monitor treatment resistance, because of its sensitiv- ity and specificity, but it really needs reproducible and standardized methods, both for the extraction and for its analyses. Regarding the mutation analysis of cftDNA, a large number of technologies is now available to analyze mutations in cftDNA, including automatic sequencing, real-time polymerase chain reaction (PCR) platforms, mass spectrometry (MS) genotyping, ampli cation proto- cols with magnetic beads in oil emulsions [beads, emul- sion, ampli cation and magnetics (BEAMing)] and next- generation sequencing (NGS), digital PCR platforms [21–25]. The sensitivity range of the available techniques Fig. 1 A ROC curve was drawn, and the area under the curve was calculated. The area under the curve for CEA was 0.567 (95% CI: 0.476 ~ 0.657, p < 0.001). When the CEA cut-off was 3.4 ng/ml, the sensitivity was 69.6% and the specificity was 48.8% molecular analyses of circulating tumor cells obtained from the peripheral blood of patients with lung cancer was useful for monitoring changes in epithelial tumor genotype during the course of treatment. However, this type of molecular analysis can be difficult due to the re- quirement of a specific, microfluidic-based device - the CTC chip. Moreover, there are approximately 486 types of EGFR-TKI domain mutations across 87 species, and new mutations are continually being identified [15, 16]. Recently, the attention moved to the possibility of isola- tion and analysis of cell-free tumor DNA (cftDNA) that, to date, represents the best candidate for identification and monitoring of molecular tumor-related alterations in blood of patients with cancer [17]. Circulating DNA Table 4 Correlation between CEA levels and disease progression B SE Wald df Sig. Exp(B) 95.0% CI for Exp(B) Lower Upper Step 1 CEA 2.007 .434 21.377 1 .000 7.443 3.178 17.428 Step 2 Age .701 .355 3.908 1 .048 2.017 1.006 4.043 CEA 2.041 .444 21.147 1 .000 7.695 3.225 18.361 Step 3 Stage .855 .428 3.997 1 .046 2.351 1.017 5.435 Age .814 .361 5.084 1 .024 2.257 1.112 4.580 CEA 1.752 .456 14.754 1 .000 5.769 2.359 14.108 According to the Cox regression analysis, CEA levels 16.2 times higher than normal at any point during the 2 months prior to the determination of progress represented an early response to disease progression Table 4 Correlation between CEA levels and disease progression Gao et al. Consent for publication Not applicable. Consent for publication Not applicable. Consent for publication Not applicable. Abbreviations CEA: Serum carcinoembryonic antigen; cftDNA: cell-free tumor DNA; CT: Computed tomography; EGFR: Epidermal growth factor receptor; EGFR- TKI: Epidermal growth factor receptor tyrosine kinase inhibitor; NSCLC: Non- small cell lung cancer; RT-PCR: Real-time quantitative PCR Conclusion Patients with elevated serum CEA levels responded more positively to EGFR-TKIs, and lung adenocarcin- oma patients with high serological CEA levels exhibited a higher rate of EGFR mutations. Received: 14 January 2016 Accepted: 6 July 2017 Received: 14 January 2016 Accepted: 6 July 2017 Funding This work was primarily supported by the Shandong Province Key Research and Development Program of China (Project No. 2016GSF201157) and National Natural Science Foundation of China (NSFC, Project NO.81602031). Availability of data and materials The datasets generated and analysed during the current study are not publicly available due this study is undergoing further research but are available from the corresponding author on reasonable request. To our knowledge, the clinical assessment of lung can- cer treatment uses the RECIST criteria as the gold stand- ard for response evaluations. However, early diagnostic CT scans for response evaluations in patients receiving EGFR-TKI therapies have severe limitations. EGFR-TKI therapy is expected to induce a response via cytostasis, rather than an objective morphologic response. The RECIST criteria are further confounded by structural ab- normalities, both before and after treatment, which may not actually be tumors. TKIs CEA serum levels may identify patients with a high risk of metastasis development prior to CT scans. Other cell adhesion molecular markers associated with lymph node metastasis, such as the chemokine receptors CCR7, CXCR3 and CCL21, could be related to distant metastasis development. Thus, studies of their associ- ation with distant metastasis development are justified. I t d th OS i t d f t li In our study, the OS-associated factors were age, clin- ical stage, and serum CEA levels. In many neoplasms, a high serum CEA level predicts residual disease or tumor relapse in patients without normal-range serum levels Gao et al. BMC Cancer (2017) 17:484 Gao et al. BMC Cancer (2017) 17:484 Page 6 of 7 Page 6 of 7 after surgery [40]. In fact, Iwasaki et al. proposed a for- mula to evaluate mortality risk based on CEA serum levels, histological type, and the presence of positive me- diastinal lymph nodes [41] High CEA serum levels may reflect micrometastatic disease, although we detected no differences in the CEA serum levels between patients of different clinical stages. This observation suggests that the prognostic role of high CEA serum levels may be completely accounted for by tumor change. CEA repre- sents an important tumor marker associated with several physiopathological CEA expression is induced by hyp- oxia inducible factor α (HIF-α), suggesting that CEA plays an important role as a micro-environmental factor during tumorigenesis and confers a worse prognosis. Author details 1 1Department of Thoracic Surgery, Shandong Provincial Hospital affiliated to Shandong University, Jinan, Shandong Province 250117, China. 2Department of Thoracic Surgery, Shandong Tumor Hospital and Institute, Jinan, Shandong Province 250117, China. 3Department of Medical Oncology, Shandong Tumor Hospital and Institute, Jinan, Shandong Province 250117, China. 4Department of Thoracic Surgery, Shandong Cancer Hospital and Institute, Jiyan Rd. 440, Jinan, Shandong 250117, People’s Republic of China. Competing interest Th h d l h Competing interest The authors declare that they have no competing interest. The authors declare that they have no competing interest. Authors’ contributions PPS and YG designed and performed experiments and wrote the manuscript; HJand BJZ provided patient material; HL helped to collect samples; PPS approved for the final vesion and submittion. All authors have read and approved the manuscript. 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. Ethics approval and consent to participate This study was approved by the medical ethics committee Shandong Tumo Hospital and Institute, the China (SDTHEC20130501). All patients will be provided with written information in the form of a Patient Information Folder, including the study aims, randomization procedure, possible AEs, and their rights and responsibilities. Written informed consent must be obtained for all patients included in the study before they are randomized in the study. The limitations of this study should be acknowledged. There is no consensus regarding the optimal timing for performing either CT scans or serum CEA measurements during or after prolonged treatments. According to RECIST version 1.1, the best radiologic response evalu- ation can be obtained at least 4 weeks after the initiation of therapy. In our study, we performed CT scans every 2 weeks after the initiation of therapy. Therefore, the rela- tively small number of patients exhibiting a radiologic re- sponse could be explained by the timing of the CT scans. 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https://openalex.org/W2166728624	https://www.scielo.br/j/rbccv/a/h8MHXBCPZTKSfVYyvNNRWkN/?lang=pt&format=pdf	Portuguese	null	Tratamento cirúrgico da síndrome da veia cava superior causado por timoma invasivo	Brazilian Journal of Cardiovascular Surgery	2,010	cc-by	2,094	1. Doutorado; Cirurgião Cardiovascular do Hospital Sugisawa; Coordenador do Serviço de Cirurgia Cardiovascular. 2. Cirurgião Oncológico do Hospital Sugisawa; Coordenador do Serviço de Oncologia do Hospital Sugisawa. 3. Cardiologista; Coordenador do Serviço de Cardiologia do Hospital Sugisawa. 4. Intensivista e Cardiologista; Responsável pela Unidade de Terapia Intensiva do Hospital Sugisawa. Abstract Resumo Paciente do sexo masculino, branco, 57 anos, portador de síndrome da veia cava superior havia 3 meses, devido a timoma invasivo em mediastino médio e anterior, levando a comprometimento da veia cava superior intrínseca e extrinsecamente. Após avaliação por tomografia computadorizada e angiorressonância magnética de tórax, o paciente foi submetido à ressecção radical do timoma – derivação venosa da veia subclávia esquerda para átrio direito, com tubo de PTFE (politetrafluoroetileno). Relevante caso de timoma invasivo ocasionando a oclusão da veia cava superior. A evolução clínica, após 7 meses, foi considerada satisfatória. We report on a case of a 57 years-old white male, patient, who presented superior vena cava syndrome (SVC) for 3 months, derived from an invasive thymoma in the medium and anterior mediastinum, compromising intrinsic and extrinsic to the SVC. After evaluation by computed tomography and magnetic angioresonance of the thorax, the patient underwent radical resection of the thymoma – bypass from left subclavian vein to right atrium, using polytetrafluoroethylene tube. Relevant case of invasive thymoma causing the occlusion of SVC. The clinic evolution of the patient after 7 months was considered satisfactory. Descritores: Síndrome da veia cava superior. Neoplasias do timo. Timoma. Descriptors: Superior vena cava syndrome. Thymus neoplasms. Thymoma. Endereço para correspondência: Dr. George Ronald Soncini da Rosa Av. Iguaçu,1236 salas 314-315 – Curitiba, PR, Brasil – CEP 80250-190. E-mail: soncini@bighost.com.br Rev Bras Cir Cardiovasc 2010; 25(2): 257-260 Rev Bras Cir Cardiovasc 2010; 25(2): 257-260 RELATO DE CASO Trabalho realizado no Hospital Sugisawa, Curitiba, PR, Brasil. Artigo recebido em 20 de julho de 2009 Artigo aprovado em 13 de novembro de 2009 RBCCV 44205-1184 RBCCV 44205-1184 urgical treatment of the superior vena cava syndrome caused by invasive thymom eorge Ronald SONCINI DA ROSA1, Ney TAKIZAWA2, Douglas SCHIMIDT3, Mitur SUG Trabalho realizado no Hospital Sugisawa, Curitiba, PR, Brasil. Endereço para correspondência: Dr. George Ronald Soncini da Rosa Av. Iguaçu,1236 salas 314-315 – Curitiba, PR, Brasil – CEP 80250-190. E-mail: soncini@bighost.com.br Artigo recebido em 20 de julho de 2009 Artigo aprovado em 13 de novembro de 2009 INTRODUÇÃO vasos, encontra-se um grande número de pacientes – portadores desta enfermidade – que não apresentam condições de enfrentar uma ressecção cirúrgica, por dificuldades técnicas ou por apresentarem morbi- mortalidade alta para o procedimento e/ou baixo impacto na sobrevida. Embora poucos pacientes concordem com o tratamento paliativo com a radioterapia e a quimioterapia, muitos dos tumores torácicos são de mau prognóstico [2]. Há um subgrupo de pacientes que apresenta risco de vida imediato, ocasionado pela insuficiência cardíaca A síndrome da veia cava superior (VCS) é incapacitante e potencialmente ameaçadora, consequência de uma complicação neoplásica ou doença inflamatória do mediastino. Neste cenário, as neoplasias malignas mediastinais figuram como uma das principais causas da síndrome da VCS [1]. A despeito da maior incidência de mortalidade causada por tumores torácicos envolvendo o coração e grandes 257 Rev Bras Cir Cardiovasc 2010; 25(2): 257-260 Após o tratamento radioterápico e já transcorridos 5 anos, uma tomografia de tórax revelou massa mediastinal envolvendo a veia cava superior e a veia braquiocefálica direita, tanto externa quanto internamente. congestiva, pela embolização sistêmica e/ou colapso cardiovascular. A terapia paliativa é inefetiva e a terapia cirúrgica radical é a única opção [2]. A ressecção radical e reconstrução da veia cava superior com uso de circulação extracorpórea (CEC) é mandatória nos casos de timomas avançados [3], embora a CEC seja raramente necessária na ressecção do timoma não invasivo. A ressonância magnética do tórax e a angiorressonância da aorta torácica revelaram massa discretamente heterogênea, localizada na região do mediastino médio e anterior, envolvendo as veias braquiocefálica direita e cava superior, sem fluxo, com opacificação da veia cava superior (VCS) a partir da altura da confluência da veia ázigo (Figura 1). O timoma é uma afecção rara, embora referido como o tumor mais frequente do mediastino anterior, compreendendo aproximadamente 20% a 30% das massas mediastinais em adultos. Ocorre principalmente na 5ª e 6ª década da vida e dois tipos são descritos: o não invasivo, encapsulado (cerca de 2/3 dos casos) e o invasivo [4]. O que realmente define sua malignidade é o achado cirúrgico: a) invasão local (pulmão, pleura, pericárdio, grandes vasos, diafragma); b) metástases intra ou extratorácicas. INTRODUÇÃO O tratamento cirúrgico foi indicado e, com o devido consentimento, o paciente foi submetido à ressecção completa das veias cava superior e braquiocefálica direita e parte da esquerda, com realização de uma derivação venosa com tubo de politetrafluoroetileno (PTFE) 8 mm da veia subclávia esquerda para o átrio direito (Figura 2). A canulação arterial foi realizada no arco aórtico e a drenagem venosa pela veia cava inferior com o auxílio da circulação extracorpórea (CEC). SONCINI DA ROSA, GR ET AL - Tratamento cirúrgico da síndrome da veia cava superior causado por timoma invasivo RELATO DE CASO Paciente do sexo masculino, branco, 57 anos, com sinais e sintomas de síndrome da veia cava superior (VCS), apresentava ao exame um importante ingurgitamento das veias jugulares, edema de face, pescoço e parede anterior do tórax, ptose palpebral esquerda, dificuldade de deglutição e respiratória, com evolução de aproximadamente 3 meses. O mesmo paciente já se submetera a uma timectomia parcial em outro serviço, cujo laudo imunohistoquímico teve diagnóstico de timoma B3 (carcinoma tímico bem diferenciado), sendo seguido com tratamento por radioterapia externa. A CEC foi iniciada a partir da ressecção do tumor no átrio direito, para que houvesse melhor drenagem venosa e isolamento do segmento proximal ao átrio direito. Neste átrio, evidenciou-se a presença de tecido tumoral móvel, ressecado, com a necessidade de pinçamento parcial do mesmo, mas sem exigir a utilização de solução cardioplégica. O tempo da CEC foi de 35 minutos. A análise histológica da peça cirúrgica teve o diagnóstico de timoma B3 (carcinoma tímico bem diferenciado). Fig. 1 - Imagens da angioressonância venosa e arterial dos vasos torácicos. As setas indicam a oclusão da veia cava superior e inominada direita causada por uma massa discretamente heterogênea localizada na região do mediastino médio e anterior Fig. 1 - Imagens da angioressonância venosa e arterial dos vasos torácicos. As setas indicam a oclusão da veia cava superior e inominada direita causada por uma massa discretamente heterogênea localizada na região do mediastino médio e anterior 258 Rev Bras Cir Cardiovasc 2010; 25(2): 257-260 SONCINI DA ROSA, GR ET AL - Tratamento cirúrgico da síndrome da veia cava superior causado por timoma invasivo Fig. 2 - Na imagem A, está representada aparência macroscópica do tumor após retirada em bloco com os vasos envolvidos. Na imagem B, a seta indica a derivação venosa, veia subclávia esquerda e átrio direito realizada com tubo de PTFE 8 mm Fig. 2 - Na imagem A, está representada aparência macroscópica do tumor após retirada em bloco com os vasos envolvidos. Na imagem B, a seta indica a derivação venosa, veia subclávia esquerda e átrio direito realizada com tubo de PTFE 8 mm No 5º dia pós-operatório, evoluiu com miastenia gravis, sendo tratado com piridostigmina (Mestinon®) e prednisona. Após a retirada dos drenos torácicos, o paciente foi anticoagulado com varfarina sódica (mantendo o controle do TAP mensalmente). RELATO DE CASO A partir deste caso, percebemos que pequenos timomas do mediastino anterior se infiltraram na veia braquiocefálica e cresceram ao longo do fluxo venoso dentro da veia cava superior, para o átrio direito, como pólipos [7]. Há divergências na literatura quanto à melhor forma de estadiar e classificar essa doença. Os métodos mais importantes são de Masaoka et al., que determina o grau de envolvimento da doença, clínica e histopatologicamente, e do French Study Group on Thymic Tumors (sistema GETT), que é baseado em aspectos cirúrgicos e patológicos do tumor [5]. O paciente recebeu alta hospitalar após o 15º dia de internamento para realizar quimioterapia ambulatorial. A observação pós-operatória foi realizada por um período de 7 meses. Fato importante, neste caso, foi a ocorrência da oclusão das veias cava superior e braquiocefálica direita causado por timoma invasivo, sem encontrar na literatura científica ou em citações da RBCCV, relato de caso semelhante. Ressecção total seguida por radioterapia é o tratamento de escolha para todos os timomas, exceto tumores em estágio IA (de Masaoka) [5], que podem ser tratados somente com cirurgia. Embora o timoma seja radiossensível, há controvérsias quanto à aplicação de radioterapia em pacientes com timoma invasivo. DISCUSSÃO A síndrome VCS é normalmente associada com malignidade e mau prognóstico [5]. Esses casos incluem câncer de pulmão em estados avançados (57%), tumores mediastinais (20%), e metástases sólidas (5%) [4]. Timoma que leve à síndrome da VCS é raro (4%), e a causa mais comum é a compressão extrínseca [4]. Na literatura científica internacional, são encontrados apenas nove casos do crescimento intracaval atingindo a síndrome da VCS [6]. A maior parte dos pacientes com a síndrome da VCS causada por tumores malignos é tratada com radioterapia e/ou quimioterapia como tratamento paliativo, e possuem uma média de sobrevida de 3 meses [4]. Stents radiológicos constituem técnica segura e oferecem rápida paliação, mas a ressecção radical em tumores que levam à obstrução e à infiltração da VCS e veias braquiocefálicas é um tratamento 259 Rev Bras Cir Cardiovasc 2010; 25(2): 257-260 3. Nakahara K, Ohno K, Hashimoto J, Maeda H, Miyoshi S, Sakurai M, et al. Thymoma: results with complete resection and adjuvant postoperative irradiation in 141 consecutive patients. J Thorac Cardiovasc Surg. 1988;95(6):1041-7. raro, como observado neste estudo. Devido à existência de oclusão total da veia cava superior por crescimento intraluminal de tecido tumoral chegando até o átrio direito, o uso de stents não foi possível, fator que possibilitou a terapêutica endovascular. 4. Kallás E, Hueb AC, Kallás IE, Kallás AC. Timoma do mediastino médio: relato de caso. Rev Bras Cir Cardiovasc. 2005;20(2):189-91. No tratamento do timoma, a cirurgia é considerada a terapia principal. Recentemente, várias terapias que incluem cirurgia, radioterapia e quimioterapia estão sendo utilizadas nos casos de timomas invasivos [8-11]. A associação do timoma com miastenia gravis ocorre em 50% dos pacientes. Por outro lado, apenas 15% dos pacientes com miastenia gravis apresentam timoma [4]. Neste caso, mesmo removendo o timoma, o paciente apresentou quadro clínico de miastenia gravis. 5. Pêgo-Fernandes PM, Ebaid GX, Galizia MS, Marchiori P, Suso FV, Jatene FB. Timoma: discussão sobre tratamento e prognóstico. J Pneumol. 2001;27(6):289-94. 6. Konstantinov IE, Saxena P, Koniuszko M, Ghosh S, Low VH, Khor TS, et al. Superior vena cava obstruction by tumour thrombus in invasive thymoma: diagnosis and surgical management. Heart Lung Circ. 2007;16(6):462-4. No caso relatado, optamos pela cirurgia radical, com a realização da derivação da veia subclávia esquerda para átrio direito, pois os sintomas consequentes da síndrome da veia cava superior já referidos levaram o paciente a uma péssima qualidade de vida. DISCUSSÃO Após realização da cirurgia e restabelecida a drenagem venosa superior, o paciente voltou a ganhar peso e a realizar seus afazeres cotidianos, sem dificuldades e com sobrevida de 7 meses satisfatória, livre de complicações clínicas graves. 7. Funakoshi Y, Ohta M, Maeda H, Matsuda H. Extended operation for invasive thymoma with intracaval and intracardiac extension. Eur J Cardiothorac Surg. 2003;24(2):331-3. 8. Minato N, Rikitake K, Ohnishi H, Takarabe K, Ishida H. Invasive thymoma with intracaval growth extending and directly invading the right atrium. J Cardiovasc Surg (Torino). 1999;40(6):915-7. 9. Gleeson RE, Price R, Nicholson AG, Pastorino U. Invasive thymoma extending to the right atrium: excision without cardiopulmonary bypass. J Cardiovasc Surg (Torino). 1997;38(4):429-31. SONCINI DA ROSA, GR ET AL - Tratamento cirúrgico da síndrome da veia cava superior causado por timoma invasivo 2. Park BJ, Bacchetta M, Bains MS, Downey RJ, Flores R, Rusch VW, et al. Surgical management of thoracic malignancies invading the heart or great vessels. Ann Thorac Surg. 2004;78(3):1024-30. REFERÊNCIAS 1. Dhaliwal RS, Das D, Luthra S, Singh J, Mehta S, Singh H. Management of superior vena cava syndrome by internal jugular to femoral vein bypass. Ann Thorac Surg. 2006;82(1):310-2. 10. Yokoi K, Miyazawa N, Mori K, Saito Y, Tominaga K, Imura G, et al. Invasive thymoma with intracaval growth into the right atrium. Ann Thorac Surg. 1992;53(3):507-9. 2. Park BJ, Bacchetta M, Bains MS, Downey RJ, Flores R, Rusch VW, et al. Surgical management of thoracic malignancies invading the heart or great vessels. Ann Thorac Surg. 2004;78(3):1024-30. 11. Pastorino U, Yang XN, Francese M, Durkovic S, Biglioli P. Long-term survival after salvage surgery for invasive thymoma with intracardiac extension. Tumori. 2008;94(5):772-6. 260
https://openalex.org/W2055398845	https://www.scielo.br/j/rcbc/a/Ws7szyPQKV8gJ9v8bzsxMkJ/?lang=pt&format=pdf	Portuguese	null	Formação e demanda do cirurgião pediátrico no Brasil	Revista do Colégio Brasileiro de Cirurgiões	2,009	cc-by	6,090	Trabalho realizado na Associação Brasileira de Cirurgia Pediátrica – Comissão de Ensino e Título de Especialista. 1. Cirurgiã Pediátrica do Hospital Universitário Antônio Pedro - Universidade Federal Fluminense – UFF - Niterói – RJ –BR; 2. Cirurgião Pediátrico do Hospital Geral de Pirajussara - Universidade Federal de São Paulo, SP-BR; 3. Preceptora da Residência Médica de Cirurgia Pediátrica do Hospital da Criança/ Obras Assistenciais de Irmã Dulce – BA- BR; 4. Presidente da Associação Brasileira de Cirurgia Pediátrica – SP-BR; 5. Cirurgião Pediátrico do Hospital da Criança Conceição – RS-BR; 6. Professor de Cirurgia Pediátrica Faculdade de Ciências Médicas Sta Casa de São Paulo – SP–BR; 7. Cirurgião Pediátrico do Hospital Infantil Pequeno Príncipe – PR-BR. Formação e demanda do cirurgião pediátrico no Brasil Formação e demanda do cirurgião pediátrico no Brasil Formação e demanda do cirurgião pediátrico no Brasil Formação e demanda do cirurgião pediátrico no Brasil Formação e demanda do cirurgião pediátrico no Brasil Needs and specialization for pediatric surgeons in Brazil Needs and specialization for pediatric surgeons in Brazil Needs and specialization for pediatric surgeons in Brazil Needs and specialization for pediatric surgeons in Brazil Needs and specialization for pediatric surgeons in Brazil LISIEUX EYER DE JESUS, TCBC-RJ1; ALEXANDRE SANTOS AGUIAR2; MARIA DO SOCORRO MENDONÇA DE CAMPOS3; JOSÉ ROBERTO DE SOUSA BARATELLA4; JOÃO CARLOS KETZER5; ROBERTO ANTÔNIO MASTROTI6; ANTÔNIO CARLOS M .AMARANTE7 R E S U M O R E S U M O R E S U M O R E S U M O R E S U M O Objetivo: Objetivo: Objetivo: Objetivo: Objetivo: Definir o perfil epidemiológico dos especialistas em cirurgia pediátrica no Brasil. Definir as relações mercado-oferta de trabalho em cirurgia pediátrica no Brasil. Comparar o perfil profissional do cirurgião pediátrico brasileiro ao perfil deste especialista em outros países. Métodos Métodos Métodos Métodos Métodos: Utilizando informações estatísticas fornecidas pelo IBGE, Conselho Federal de Medicina e Associação Brasileira de Cirurgia Pediátrica, definir o perfil de trabalho dos médicos especializados em cirurgia pediátrica no Brasil. Resultados: Resultados: Resultados: Resultados: Resultados: A demanda de cirurgiões pediátricos trabalhando no Brasil em horário integral é de 850 cirurgiões, caso se considere apenas o atendimento de lactentes e neonatos. Há uma centralização excessiva de cirurgiões pediátricos no sul e sudeste e falta de mão de obra nas regiões norte e nordeste. Os dados quanto ao número de cirurgiões pediátricos atuando no Brasil são conflitantes (dados de pesquisa epidemiológica da FIOCRUZ diferem de dados do CFM e da CIPE). A rotina de trabalho do cirurgião pediátrico no Brasil não é comparável com aquela dos profissionais norte-americanos e europeus, fontes da maior parte dos dados de referência em literatura. Conclusão: Conclusão: Conclusão: Conclusão: Conclusão: A demanda de cirurgiões pediátricos trabalhando no Brasil em horário integral apenas para atendimento de lactentes e neonatos é de 850 cirurgiões. Existe um desequilíbrio entre oferta e ocupação de postos de trabalho nas diversas regiões do país. O sudeste é um centro de formação de especialistas que exporta profissionais para as demais regiões do país. Os dados quanto ao número de cirurgiões pediátricos atuando no Brasil são conflitantes. er de Jesus er de Jesus er de Jesus er de Jesus er de Jesus trico no Brasil Ensino Ensino Ensino Ensino Ensino er de Jesus er de Jesus er de Jesus er de Jesus er de Jesus trico no Brasil Ensino Ensino Ensino Ensino Ensino Descritores: Descritores: Descritores: Descritores: Descritores: Cirurgia pediátrica. Demanda cirurgiões. Mercado trabalho. INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO 2009; 36(4): 356-361 Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Formação e demanda do cirurgião pediátrico no Brasil 357 yyyyy Formação e demanda do cirurgião pediátrico no Brasil 162 divididas nos anos obrigatórios de R1, R2 e R3 (forma- ção potencial de 54 cirurgiões pediatras/ano). Apenas seis vagas são oferecidas para aperfeiçoamento (R4 e R5), es- sencialmente em serviços de transplante pediátrico (fonte: CIPE/MS, ano 2007). As vagas são distribuídas geografica- mente de forma desproporcional com relação ao território (83% nos estados do Sul e Sudeste) e à população (83% das vagas distribuídas em estados da federação que detêm 68% da população), nenhuma vaga na Região Norte (Ta- bela 1). pediátricos em nosso país e é coordenada direta e inteira- mente pelo Ministério da Educação através da Comissão Nacional de Residência Médica desconsiderando, até o momento, ao menos no aspecto formal, as demandas das várias sociedades médicas. Outro problema tem sido a falta de dados que permitam determinar o contingente profissional disponível versus o que é necessário e planejar de forma correta a distribuição de vagas para formação. As vagas para resi- dência em cirurgia pediátrica têm sido oferecidas no terri- tório nacional sem obedecer a nenhum parâmetro técnico ou epidemiológico desde o reconhecimento da cirurgia pediátrica como especialidade no Brasil. Atualmente temos 70 médicos cursando residên- cia médica em cirurgia pediátrica no Brasil, distribuidos em várias etapas de formação (a taxa de ocupação das vagas oferecidas é de 43%, aproximadamente 23 cirurgiões for- mados/ano, dependendo da proporção entre R1-2-3-4-5), traduzindo um desinteresse dos cirurgiões jovens na área de cirurgia pediátrica, mercado de trabalho insuficiente e/ ou persistência de vagas na grade oficial do MS em servi- ços que deixaram de oferecer efetivamente formação na especialidade (fonte: CIPE, ano 2007). A grande maioria dos médicos em formação está nas regiões sudeste (35 residentes em São Paulo, quatro em Minas Gerais e oito no Rio de Janeiro) e região sul (dois residentes em Santa Catarina, sete no Paraná e seis no Rio Grande do Sul) (Tabela 1). Este trabalho tem o objetivo de mapear as vagas e os níveis de ocupação das residências em cirurgia pediátrica no Brasil. INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO Busca também projetar as necessidades reais de mão de obra especializada no país, a fim de possibilitar es- tratégias corretas de planejamento da formação. * 32% do total de vagas no estado de São Paulo e 22% no estado do Rio de Janeiro. MÉTODOS MÉTODOS MÉTODOS MÉTODOS MÉTODOS 1. Quantificação e mapeamento das vagas de residência médica em Cirurgia Pediátrica no Brasil e suas taxas de ocupação. Dados obtidos através da Associação Brasileira de Cirurgia Pediátrica (CIPE) e Ministério da Saú- de, ano base 2007. O número de profissionais associados à CIPE é de 851. Excluímos da análise 52 profissionais que são só- cios remidos, considerando que por sua idade possivel- mente estarão aposentados ou exercendo a especialida- de em horário restrito. No CFM estão registrados 721 médicos em atividade como especialistas em cirurgia pediátrica. Nem todos estes profissionais se dedicam ex- clusivamente ao trabalho de especialista ou trabalham em tempo integral. A taxa de afastamento anual é desconhe- cida. Os grupos de especialistas vinculados à CIPE e ao CFM se superpõem apenas parcialmente, e há profissio- nais que se declaram cirurgiões pediátricos e não estão registrados no CFM ou na CIPE, embora seja excepcional que um profissional formalmente treinado na área e apto a atuar em cirurgia pediátrica complexa não esteja regis- trado ao menos em uma das duas instituições. A conclu- são evidente é que a estimativa real dos cirurgiões exer- cendo a cirurgia pediátrica no Brasil pode ser, no máximo, aproximada. 2. Quantificação da população pediátrica brasi- leira e projeção das necessidades de atendimento cirúrgico pediátrico, considerando o número de cirurgiões pediátricos disponíveis no mercado brasileiro e os profissionais egres- sos da especialização (por ano). Dados obtidos dos censos populacionais do IBGE, CFM e FIOCRUZ. Para estimar a demanda de atendimento cirúrgi- co pediátrico buscamos quantificar as incidências das do- enças cirúrgicas mais comuns na população infantil, utili- zando índices de incidência colhidos em um tratado de Ci- rurgia Pediátrica amplamente conhecido e adotado2. Rev. Col. Bras. Cir. 2009; 36(4): 356-361 INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO INTRODUÇÃO anças mais velhas e o atendimento inicial em casos de trauma infantil (padrão habitual nos países de primeiro mundo). O O planejamento da formação de médicos especialistas para atender corretamente à demanda populacional sem que haja excesso de profissionais tem sido sempre uma preocupação nos países avançados. Em geral as associa- ções de especialistas geram estas diretrizes, em conjunto com os conselhos gestores, com atualizações permanentes a partir de revisões periódicas das demandas específicas. 2. Se o atendimento a doenças urológicas da cri- ança (em torno de 60% da demanda de atendimento em cirurgia pediátrica) será feito por cirurgiões pediátricos (caso do Brasil e alguns países europeus) ou urologistas subespecializados (caso da América do Norte). 3. Se os limites de faixa etária para atendimento pelo cirurgião pediátrico incluirão pacientes pós-puberais até 18 (caso do Brasil, conforme determinado pelo Estatuto da Criança e do Adolescente) ou 21 anos (EUA). A oferta de vagas para formação é regulada pelo equilíbrio entre demanda e disponibilidade de profissionais, por sua vez determinados por variáveis populacionais, pela demografia de profissionais e, eventualmente, pelo surgimento de novos nichos de atuação a partir de avanços tecnológicos. No caso da Cirurgia Pediátrica a demanda de profissionais depende diretamente do crescimento demográfico (número de crianças na população) e dos en- cargos específicos do especialista em cirurgia pediátrica em cada sociedade, por exemplo: 4. Se os profissionais de cirurgia pediátrica preci- sarão ou não se envolver no atendimento geral da popula- ção, limitando sua disponibilidade para a população pediátrica (caso dos países mais pobres, em especial afri- canos e asiáticos) 1. Seria esperado que os conselhos de medicina, Associação Médica Brasileira, Colégio Brasileiro de Cirurgi- ões e CIPE participassem do controle da formação de cirur- giões pediátricos no Brasil, considerando as diretrizes e pro- jeções de atendimento do Ministério da Saúde (MS) e SUS. No entanto, a oferta de vagas para formação de cirurgiões 1. Se todo o atendimento a crianças será feito por especialistas ou se estes atuarão apenas em casos com- plexos, malformações incomuns e neonatos, enquanto pro- fissionais generalistas assumem os casos de rotina em cri- 1. Se todo o atendimento a crianças será feito por especialistas ou se estes atuarão apenas em casos com- plexos, malformações incomuns e neonatos, enquanto pro- fissionais generalistas assumem os casos de rotina em cri- Rev. Col. Bras. Cir. Rev. Col. Bras. Cir. 2009; 36(4): 356-361 Tabela 1 - Tabela 1 - Tabela 1 - Tabela 1 - Tabela 1 - Distribuição de vagas para residência em Cirurgia Pediátrica e cirurgiões pediátricos atuantes no Brasil (fonte: CIPE, ano 2007, IBGE 2, CFM, julho 2008). DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO A demanda de trabalho para o cirurgião pediátrico respeita especificidades nacionais. Os cirurgiões pediátricos brasileiros atendem crianças até os 18 anos de idade (de- terminação do Estatuto da Criança e do Adolescente, lei Federal do País) e atuam em todas as doenças cirúrgicas pediátricas, à exceção de doenças oftalmológicas, otorrinolaringológicas, ortopédicas, cardíacas e neurocirúrgicas. Doenças urológicas infantis em geral, ci- rurgia torácica na infância e várias cirurgias plásticas reconstrutoras para doenças congênitas são atendidas pe- los profissionais de cirurgia pediátrica. Especialistas em ou- tras áreas cirúrgicas freqüentemente usam a ajuda dos ci- rurgiões pediátricos para atender crianças e portadores de malformações congênitas. Esta amplitude de atuação é diferente da maioria dos países europeus e, certamente, da América do Norte, e impede que as demandas de tra- balho brasileiras sejam comparadas com dados de literatu- ra quanto à atuação do cirurgião pediatra em países euro- peus e norteamericanos. 2. Demanda de cirurgia pediátrica urológica com- plexa: aproximadamente 0,5% da população pediátrica (In- cidências consideradas na estimativa: hipospádias 0,3%, estenoses de junção ureteropiélica 0,2%). 3. Demanda de cirurgia pediátrica geral comple- xa: aproximadamente 5% da população pediátrica (Inci- dências consideradas na estimativa: megacólon agangliônico 0,02%, refluxo gastroesofágico complexo ou da população de encefalopatas 5%) 4. Demanda de cirurgia neonatal: aproximada- mente 1,5 % da população pediátrica (Incidências consi- deradas na estimativa: estenose hipertrófica de piloro 0,02%, atresias intestinais 0,03%, enterocolites necrosantes 0,2% (considerando uma incidência de enterocolite de 10% numa população de 10% de prematuros a partir do total de neonatos e uma taxa de enterocolites cirúrgicas de 20% do total de casos), malformação ano-retal 0,02%, atresia de esôfago 0,03%, gastrosquise 0,02%). O estoque calculado de atendimento cirúrgico pediátrico operatório na população pediátrica é de apro- ximadamente 10% da população de crianças. Para avali- ar a demanda total de trabalho do cirurgião pediátrico deve ser acrescida a necessidade de atendimento para avaliação clínica, aconselhamento pré-natal, consultas para diagnóstico e seguimento e atendimento ao trauma pediátrico (aproximadamente 10% dos atendimentos em emergência pediátrica, atingindo 0,3% da população pediátrica/ano) 4 . Os números discriminados acima são conserva- dores, já que as crianças vitimadas pela alta taxa de morta- lidade infantil também são atendidas por cirurgiões pediátricos, em especial casos de malformação complexa e pacientes atendidos em unidades de terapia intensiva neonatal. Mesmo sem incluir estes casos a taxa anual po- tencial de cirurgias neonatais no Brasil é de 58320 cirurgi- as/ ano. RESULTADOS RESULTADOS RESULTADOS RESULTADOS RESULTADOS No ano de 2007 foram oferecidas pelo MS 168 vagas para formação de cirurgiões pediátricos no Brasil, buição de vagas para residência em Cirurgia Pediátrica e cirurgiões pediátricos atuantes no Brasil (fonte: CIPE, 007, IBGE 2, CFM, julho 2008). Tabela 1 - Tabela 1 - Tabela 1 - Tabela 1 - Tabela 1 - Distribuição de vagas para residência em Cirurgia Pediátrica e cirurgiões pediátricos atuantes no Brasil (fonte: CIPE, ano 2007, IBGE 2, CFM, julho 2008). Região Região Região Região Região População População População População População % vagas no Brasil % vagas no Brasil % vagas no Brasil % vagas no Brasil % vagas no Brasil Vagas residência Vagas residência Vagas residência Vagas residência Vagas residência Cirurgiões pediatras Cirurgiões pediatras Cirurgiões pediatras Cirurgiões pediatras Cirurgiões pediatras CIPE/ ocupação CIPE/ ocupação CIPE/ ocupação CIPE/ ocupação CIPE/ ocupação em exercício em exercício em exercício em exercício em exercício Norte 7% 0 0(inválido) 21 (3%) Nordeste 29% 12% 20 (35%) 112 (16%) Centro-oeste 6% 5% 9 (56%) 81 (11%) Sudeste 43% 67% * 112 (39%) 319 (44%) Sul 15% 16% 27 (46%) 188 (26%) Total 100% 100% 168 (43%) 721 (100%) Rev. Col. Bras. Cir. 2009; 36(4): 356-361 Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus diát i B il Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Formação e demanda do cirurgião pediátrico no Brasil 358 Eyer de Jesus Eyer de Jesus ye de Jesus ye de Jesus Eyer de Jesus Formação e demanda do cirurgião pediátrico no Brasil pediátricos/ano no Brasil, considerando turnos integrais de trabalho, apenas para atender à demanda de cirurgias para pacientes durante o primeiro ano de vida, desconsiderado o tempo de atendimentos clínicos associados aos doentes cirúrgicos e o atendimento além do primeiro ano de vida. A população brasileira é de 188298099 habitan- tes, com taxa de natalidade de 21/1000 habitantes/ano e mortalidade infantil de 28/1000 nascidos vivos3 nascem aproximadamente 4000000 crianças/ano em território na- cional. Considerando a mortalidade infantil a população é acrescida de aproximadamente 38888000 crianças/ano. 1. Demanda de cirurgia pediátrica básica: apro- ximadamente 3,5 % da população pediátrica (Incidências consideradas na estimativa: hérnias inguinais 1%, criptorquias 1%, apendicites 0,3%, necessidade de aces- sos vasculares por intervenção cirúrgica 1%). DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO 2009; 36(4): 356-361 Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus d Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Formação e demanda do cirurgião pediátrico no Brasil 359 ye de Jesus ye de Jesus yyy Formação e demanda do cirurgião pediátrico no Brasil versus cirurgiões gerais 5-6 especialmente em trauma, apen- dicite, refluxo vesicoureteral7 e estenose hipertrófica de piloro8 , com nítida vantagem para os atendimentos feitos por cirurgiões pediátricos. Alguns autores têm sugerido que na impossibilidade de obter atendimento cirúrgico especi- alizado irrestrito para crianças uma solução intermediária seria prover um treinamento básico em cirurgia pediátrica a profissionais não especializados. Problemas na atuação de cirurgiões não especialistas em atendimento pediátrico devem surgir inexoravelmente nos próximos anos, com a aposentadoria dos profissionais com formação cirúrgica mais geral treinados até os anos 70-80 do século XX, que serão substituídos por médicos sem a mesma frequência de ex- posição a casos pediátricos, considerando as modificações na formação de base dos cirurgiões gerais 9-10. O grupo de autores deste trabalho acredita que se a qualidade de aten- dimento ao paciente é melhor se feita pelo profissional es- pecializado, a solução ideal, obviamente, é aumentar a disponibilidade de profissionais para o atendimento, embo- ra isto dependa da empregabilidade e disponibilidade de vagas para formação destes cirurgiões. parte da força de trabalho está no sudeste-sul do país, de- terminando limitações no atendimento da população das demais regiões, em especial se considerarmos as grandes distâncias envolvidas, os problemas sociais e trabalhistas das famílias mais pobres (trabalho na agricultura e trabalho informal mediante pagamento puntual), a precariedade dos sistemas de transporte em várias regiões, as restrições de assistência social e as dificuldades práticas na interação entre serviços. Paradoxalmente, é comum no Brasil a não dis- ponibilidade do profissional de alta especialização para atu- ação integral na sua área específica de formação. Este pro- blema, típico de países pobres com pouca disponibilidade de médicos em geral ou de falhas de gerenciamento do trabalho médico, determina uma disponibilidade efetiva parcial dos profissionais especializados. Uma causa é a oferta proporcionalmente menor de trabalho para especialistas no serviço público de saúde (SUS), que atende a > 80% da população e é o maior empregador de médicos no país, mas emprega principalmente emergencistas e médicos atu- ando nas quatro especialidades básicas13. DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO A demanda anual de cirurgia pediátrica geral na população de 3888000 crianças/ano (taxa de nascimentos anuais sobrevivente às cifras de mortalidade infantil) tam- bém é fortemente discutível, já que consideramos aqui ex- clusivamente doenças típicas e de maior incidência na po- pulação pediátrica. Hérnias inguinais, criptorquias, hidronefroses congênitas, hipospádias, megacólon congê- nito, refluxos gastroesofágicos complexos e acessos vasculares em UTI são caracteristicamente atendidos no primeiro ano de vida. Do grupo de doenças utilizadas como padrão neste trabalho, apenas as apendicites se apresen- tariam predominantemente em outras faixas etárias. Desta forma, no mínimo 9% da população pediátrica = um ano idade necessita de assistência cirúrgica/ ano, acrescendo à demanda perinatal mais 349920 cirurgias. Esta última vertente é especialmente difícil de quantificar. O cirurgião pediátrico é o profissional com as melhores qualificações para a avaliação da criança traumatizada, como líder do time de atendimento multidisciplinar ao trauma pediátrico, mas o primeiro aten- dimento da criança traumatizada e a triagem dos níveis de gravidade freqüentemente são feitos por toda uma gama de profissionais médicos emergencistas e a grande maioria de casos de trauma pediátrico necessita principalmente de atendimento ortopédico ou neurocirúrgico. Os países avançados têm restringido o atendi- mento do cirurgião pediátrico especialista a casos de doen- ça complexa e neonatos e vêm debatendo a necessidade e a pertinência do atendimento dos casos mais simples de cirurgia em crianças por cirurgiões especializados, conside- rando os resultados das cirurgias feitas por não especialis- tas e a inexistência de especialistas em número suficiente para suprir a esta demanda em seus territórios. Vários arti- gos estão publicados com relação à qualidade de atendi- mento, custos e resultados de cirurgiões especializados Considerando como critério de atividade ideal em centro cirúrgico para um cirurgião pediátrico aproximada- mente três cirurgias/dia, quatro dias/semana, 40 semanas/ ano cada cirurgião pediátrico realizaria 480 cirurgias/ano. Apresentando-se uma demanda cirúrgica de 408240 cirur- gias seriam necessários aproximadamente 850 cirurgiões Rev. Col. Bras. Cir. DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO É bastante co- mum encontrar cirurgiões pediatras trabalhando como ci- rurgiões gerais, pediatras e/ou emergencistas mesmo em locais em há déficit de atendimento na área. Da mesma forma, é frequente que não esteja disponível a estrutura logística necessária ao trabalho do cirurgião pediátrico (UTI pediátrica, atendimento neonatal clínico, anestesia de alta complexidade). Os dados europeus e norteamericanos levam a questionar se os critérios fortemente limitadores da forma- ção de especialistas nestes países estão corretos e desta- cam a concentração de cirurgiões pediátricos em grandes centros, cidades de maior porte e centros acadêmicos, obri- gando a disponibilizar um sistema permanente e eficaz de contra-referência e transporte de crianças em nações de grande extensão territorial, e gerando absenteísmo de tra- balho dos pais, dificuldades para o seguimento pós-opera- tório em doenças de alta complexidade e necessidade de atendimento a outros filhos no caso de remoção da criança doente para locais distantes da residência da família. Conforme a tabela 1 evidencia, a distribuição de cirurgiões pediatras é inadequada no território brasileiro com relação à população: pode-se presumir demanda reprimi- da em norte e nordeste e, na prática, na região centro- oeste, exceto as cidades de Brasília e Goiânia. Nos parece evidente que esta distribuição de profissionais é determina- da pela disponibilidade de mercado, nível de remunera- ção, estruturação de serviços de apoio e qualidade de vida oferecidos aos profissionais. Em trabalho apresentado no XXIX Congresso Brasileiro de Cirurgia Pediátrica Aguiar et al demonstraram que a distribuição de cirurgiões pediátricos no país é paralela à presença de grupos de medicina priva- da financiadores da saúde na população14. Os EUA dispõem de 33 centros de treinamento em cirurgia pediátrica. Apesar das projeções relativamente pessimistas de McNeill et al de que haveria um excesso de cirurgiões pediátricos no país a partir do século XX9 um trabalho recente cataloga cerca de 800 cirurgiões pediátricos em atividade naquele país, com uma deficiência atual de cerca de 200 profissionais e uma queda preocupante no contingente de profissionais jovens interessados na área10 . A crise de formação e disponibilidade de profissionais em cirurgia tem sido tão grave que em alguns países pacientes pediátricos têm sido delegados até a profissionais não mé- dicos, em razão da disponibilidade limitada de especialis- tas para atendimento: na Inglaterra, por exemplo, postectomias pediátricas têm sido executadas por enfer- meiras11 . Visivelmente o sudeste é um centro formador, notadamente São Paulo. DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO Uma proporção grande dos pro- fissionais formados nesta região migra ou retorna a seus estados de origem para exercer a especialidade. A crise mundial de formação de profissionais médicos, em especial na área de cirurgia geral e especi- alizada, tem levado a uma escassez de cirurgiões, com projeções pessimistas para um futuro próximo15. A tendên- cia é de estimular a formação, criar e prover uma nova logística no exercício da profissão, respeitando as deman- das de qualidade de vida e disponibilidade para atendi- mento familiar. A profissão de médico tem uma participa- ção progressivamente maior do sexo feminino e os profissi- onais jovens se inserem num momento social coerente com estas aspirações16. No Brasil metade dos estudantes de medicina é do sexo feminino e originária de famílias com renda familiar elevada17 .Considera-se que é imperativo Os problemas sociais e familiares causados pela espera longa por procedimentos pediátricos são evidentes. Mais de 80% dos pais canadenses aguardando cirurgias não-urgentes para seus filhos julgam uma demora maior que 3 meses inaceitável e 94% referem grandes doses de estresse familiar nesta situação. Mais de 61% dos doentes envolvidos nesta pesquisa já esperavam por mais de 6 meses pela cirurgia12 . No Brasil a centralização excessiva do atendimen- to e formação em cirurgia pediátrica é evidente. A maior Rev. Col. Bras. Cir. 2009; 36(4): 356-361 Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus d l Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus 360 Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Formação e demanda do cirurgião pediátrico no Brasil Formação e demanda do cirurgião pediátrico no Brasil realimentar o interesse dos profissionais jovens nas áreas de cirurgia de alta complexidade, ainda que para isto seja necessário rever os dogmas da prática profissional. Propos- tas de emprego em carga horária parcial e estímulos sala- riais à especialização em áreas carentes são coerentes com estas tendências. Outro aspecto a ser discutido é a pequena oferta para aperfeiçoamento. É necessário discutir a demanda por mais vagas em território nacional, em especial nas áreas de urologia, traumatopediatria, transplantes, cirurgia mini- mamente invasiva e cirurgia oncológica pediátrica. DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO Este número seria acrescido de cerca de 350 profissionais em 2007, considerando as taxas atuais de for- mação, embora a determinação final do número de cirur- giões atuantes dependa do reconhecimento do quantitati- vo de profissionais afastados por aposentadoria ou remanejamento voluntário da carreira. Existe uma discordância entre os dados de profissionais registrados nesta pesquisa e o número de profissionais registrados como ci- rurgiões pediátricos no Conselho Federal de Medicina e na Associação Brasileira de Cirurgia Pediátrica, que talvez se deva à não obrigatoriedade legal de título de especialista ou vinculação com sociedades de especialidade para o exer- cício de especialidades no Brasil. Uma grande proporção dos profissionais que se auto-identificam como especialis- tas presumivelmente, não teve formação de especialidade completa em instituições oficialmente reconhecidas. Em- bora não tenhamos podido obter comprovação deste dado, dificilmente estes profissionais atuam em áreas de alta com- plexidade. O Brasil forma atualmente 32 cirurgiões pediátricos Este trabalho apresenta algumas limitações sé- rias. rias. rias. 1. Não foi computada a carga de trabalho de um cirurgião pediatra usada em atendimento não operatório, que é extremamente difícil de medir, embora tenhamos tentado considerar esta variável calculando a atuação ci- rúrgica quatro dias por semana (considerando o 5o dia útil como reservado para consultas, pareceres, plantão presencial ou sobreaviso) e tenhamos utilizado a idéia de que o ato operatório é absolutamente exclusivo do cirur- gião, enquanto que atendimentos clínicos para seguimen- to e pareceres podem ser auxiliados por outros profissionais médicos. 2. O exercício “ideal” de três cirurgias/dia/cirur- gião depende de variáveis logísticas nos centros cirúrgicos e do nível de complexidade das cirurgias e atos anestésicos propostos, variando entre os centros de atendimento (alta, baixa e média complexidade). 3. Nossos cálculos estão limitados às incidências médias apenas de doenças de alta incidência e/ou especí- ficas da população pediátrica, obtidos a partir de literatura internacional. Esta aproximação conservadora tende a su- bestimar a necessidade de cirurgiões pediátricos para aten- dimento da população infantil e pode apresentar erros caso a incidência diferencial de alguma das doenças seja subs- tancialmente diferente na população brasileira. 4. DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO A partir do ano de 2009 a CIPE passou a oferecer cursos de aperfei- çoamento em áreas específicas, nas áreas de cirurgia urológica, neonatal e atendimento a queimados, apenas para cirurgiões pediátricos e em centros quaternários de atendimento, além das vagas de formação em medicina de transplantes. A qualificação de profissionais é importan- te para fomentar núcleos de pesquisa e atualização per- manente aptos a implementar e difundir os progressos da especialidade para os centros de menor sofisticação e enti- dades primariamente assistenciais (educação continuada). Alternativamente, programas de intercâmbio sistemático de profissionais entre centros nacionais ou com centros in- ternacionais poderiam resolver o problema da não-unifor- mização da qualidade de atendimento. O número de candidatos à formação em cirurgia pediátrica diminuiu a cerca de metade nos últimos 5 anos da década de 90 nos EUA18 . No Brasil a crise na formação de cirurgiões de forma geral também é séria19, o que talvez também esteja sendo sinalizado neste trabalho, com prati- camente metade das vagas para residência em cirurgia pediátrica ociosas. Nos EUA o cálculo utilizado de um cirurgião pediátrico:100000 a 200000 crianças (< 21 anos) tem sido considerado ideal, embora seja discutível, já que a base de cálculo é a demanda apenas para cirurgias “especialista- específicas”, não considerando qualquer tempo para ativi- dade não operatória do cirurgião18,20. As taxas européias de distribuição de cirurgiões pediátricos/ população (n:100000 população total) em 1995 variavam entre 0,09 (Inglaterra) e 0,8 (Finlândia e Suíça), sugerindo que há uma distribui- ção deficitária dos profissionais em todo o mundo13. Segun- do dados do IBGE a população brasileira até os 19 anos de idade é de aproximadamente 70 milhões, que exigiria apro- ximadamente 3000 e 6000 cirurgiões pediátricos atuantes, considerando os padrões estadunidenses. Este número ates- ta a natureza extremamente conservadora dos cálculos que utilizamos na seção material e métodos, propositalmente voltados à avaliação da demanda profissional para o grupo mais específico de lactentes e neonatos. A pesquisa “Perfil dos Médicos do Brasil” detectou (em 1995) 2419 médicos que se autodeclararam cirurgiões pediátricos atuantes, dis- tribuídos entre as regiões norte, nordeste, sudeste, sul e centro oeste (respectivamente 64, 302, 1665, 282 e 106 profissionais). REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS determinante demográfico da concentração de cirurgiões pediátricos. Arch Pediatr Surg. 2009; 1(1):12-3. 1. Ameh EA, Adejuyigbe O, Nmadu PT. Pediatric surgery in Nigeria. J Pediatr Surg. 2006; 41(3):542-6. Epub 2006 Jan 31. 1. Ameh EA, Adejuyigbe O, Nmadu PT. Pediatric surgery in Nigeria. J Pediatr Surg. 2006; 41(3):542-6. Epub 2006 Jan 31. 15. Neuhaus P. Why should young doctors choose to become surgeons? Ann Surg. 2007; 246(6):911-5. 16. Smith NP, Dykes EH, Youngson GS, Losty PD. Is the grass greener? A survey of female pediatric surgeons in the United Kingdon. J Pediatr Surg. 2006; 41(11):1879-81. 2. O’Neill JA, Rowe MI, Grosfeld JL, Fonkalsrud EW, Coran AG, editors. Pediatric Surgery. 5th ed. Philadelphia: Mosby Year Book; 1998. 3. IBGE.org [Internet]. Brasil: Instituto Brasileiro de Geografia e Esta- tística. [acessado julho 2008]. Disponível em: www.ibge.gov.br 17. Ferreira RA, Peret Filho LA, Goulart EM, Valadão MM. [Undergraduate students of “Universidade Federal de Minas Ge- rais”: profile and trends]. Rev Assoc Med Bras. 2000; 46(3):224-31. Epub 2000 Nov 16. 4. Aguiar AS, Andrade A. Estudos da Comissão de Epidemiologia do Hospital Geral de Pirajussara em epidemiologia do trauma pediátrico nos municípios de Embu das Artes e Taboão da Serra, Estado de São Paulo. Dados não publicados. 18. O’Neill JA Jr, Gautam S, Geiger JD, Ein SH, Holder TM, Bloss RS, Krummel TM. A longitudinal analysis of the pediatric surgery workforce. Ann Surg. 2000; 232(3):442-53. 5. Snow BW, Cartwright PC, Young MD. Does surgical subspecialization in pediatrics provide high-quality, cost-effective patient care? Pediatrics. 1996; 97(1):14-7. 19. Jesus LE. Ensinar cirurgia: como e para quem? Rev Col Bras Cir. 2008; 35(2):136-40. 6. Mayor S. Paediatric surgery should be performed by specialist surgeons. BMJ. 2000; 320(7247):1423. 20. O’Neill JA Jr, Cnaan A, Altman RP, Donahoe PK, Holder TM et al. Update on the analysis of the need for pediatric surgeons in the United States. J Pediatr Surg. 1995; 30(2):204-10; discussion 211- 3. 7. Snow BW. Does surgical specialty care come with a higher post? Curr Opin Pediatr. 2005, 17(3):407-8. 8. Brain AJ, Roberts DS. Who should treat pyloric stenosis: the gene- ral or specialist pediatric surgeon? J Pediatr Surg. 1996, 31(11):1535-7. 21. Grosfeld JL. World Federation of Associations of Pediatric Surgeons. Declaration of pediatric surgery. J Pediatr Surg. 2001; 36(12):1743. 21. Grosfeld JL. World Federation of Associations of Pediatric Surgeons. Declaration of pediatric surgery. J Pediatr Surg. 2001; 36(12):1743. 9. Agradecimentos Agradecimentos Agradecimentos Agradecimentos Agradecimentos Agradecemos ao colega Edevard Araújo pela grande ajuda para a coleta de dados e como crítico e estimulador deste trabalho. A B S T R A C T A B S T R A C T A B S T R A C T A B S T R A C T A B S T R A C T Objective: Objective: Objective: Objective: Objective: To define the distribution of Pediatric Surgeons in Brazil and the distribution of job offered for specialists in the country. To compare the professional profile of Brazilian Pediatric Surgeons with other countries. Methods: Methods: Methods: Methods: Methods: Statistical data derived from Brazilian Institute of Geography and Statistics (IBGE), Brazilian Medical Council (CFM) and Brazilian Society of Pediatric Surgery (CIPE) were used to define the professional profile of pediatric surgeons in Brazil. Results: Results: Results: Results: Results: Eight-hundred-and-five pediatric surgeons are needed in Brazil only to treat neonates and toddlers. Professionals are excessively concentrated in South and Southeast and insufficient numbers are found in North and Northeast. Data about the number of pediatric surgeons working in Brazil are conflicting: data obtained from FIOCRUZ differ from those from CFM and CIPE. The working routine of Brazilian pediatric surgeons is different from that of North-American and European specialists, from where the major part of our references is extracted. Conclusion Conclusion Conclusion Conclusion Conclusion: Brazil needs a minimum of 805 pediatric surgeons working full time only to treat nenonates and toddlers. Specialists are not well distributed throughout the country. The southeast is a training center and exports specialists to the rest of the country. Epidemiological data concerning the distribution of professionals in Brazil are conflicting. DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO DISCUSSÃO Certamente há profissionais especializados trabalhando como cirurgiões pediátricos sem registro na CIPE ou no CFM, em virtude de não haver exigência legal a respeito: dependendo da proporção destes profissionais com relação aos registrados e, inegavelmente, da com- plexidade dos procedimentos que possam exercer, pode- mos ter superestimado a necessidade de profissionais, embora isto seja fortemente improvável, principalmente no que concerne às cirurgias neonatais ou de alta com- plexidade. O Brasil forma atualmente 32 cirurgiões pediátricos por ano. Dados dos EUA sugerem que cerca de 30 novos cirurgiões ingressam por ano no mercado profissional ame- ricano10 . 5. A metodologia proposta aqui para os cálculos é, até o momento, inédita em literatura, e a crítica metodológica é necessária. 5. A metodologia proposta aqui para os cálculos é, até o momento, inédita em literatura, e a crítica metodológica é necessária. Rev. Col. Bras. Cir. 2009; 36(4): 356-361 Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus F ã d Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Eyer de Jesus Formação e demanda do cirurgião pediátrico no Brasil 361 yyyyy Formação e demanda do cirurgião pediátrico no Brasil Há flagrantes desequilíbrios na distribuição de cirurgiões pediátricos no Brasil. É necessário um esforço de integração entre a CIPE, a AMB, o CBC, o CFM e os Minis- térios da Educação e da Saúde no sentido de equilibrar melhor demanda de trabalho, distribuição de postos de tra- balho e vagas para formação de especialistas a partir de parâmetros epidemiológicos, naturalmente, provendo uma estrutura de trabalho suficiente para atrair e empregar um cirurgião pediátrico nas regiões onde exista uma demanda reprimida. Afinal, atender ao estabelecido no protocolo de Kyoto 21 é o objetivo principal e final do trabalho do cirur- gião pediátrico: uma criança tem necessidades específicas, do ponto de vista psicológico, metabólico e anatômico, e toda criança doente tem o direito de ser tratada num ambi- ente apropriado por profissionais adequadamente treina- dos para obter os melhores resultados, com um mínimo de seqüelas físicas e mentais. Key words Key words Key words Key words Key words: Pediatric surgery. Surgery workforce. Medical job market. REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS REFERÊNCIAS O’Donovan A, Varadaraj H, Delaney PV, Flood H, Drumm J, Grace PA. The future of paediatric surgery outside specialist paediatric hospitals. Ir J Med Sci. 2001; 170(4):228-30. Aceito para publicação em 05/01/2009 Conflito de interesse: nenhum 10. Nakayama DK, Newman KD. Pediatric surgery workforce: population and economic issues. J Pediatr Surg. 2008; 43(8):1426- 31; discussion 1432. Fonte de financiamento: nenhuma Como citar este artigo: Como citar este artigo: Como citar este artigo: Como citar este artigo: Como citar este artigo: 11. Palit V, Menebhi DK, Taylor I, Young M, Elmasry Y, Shah T. A unique service in UK delivering Plastibell circumcision: review of 9- year results. Pediatr Surg Int. 2007; 23(1):45-8. Epub 2006 Oct 6. ggggg Jesus LE, Aguiar AS, Campos MSM, Baratella JRS, Ketzer JC, Mastroti RA, Amarante ACM. Formação e demanda do cirurgião pediátrico no Brasil. Rev Col Bras Cir. [periódico na Internet] 2009; 36(4). Disponível em URL: http://www.scielo.br/rcbc ggggg Jesus LE, Aguiar AS, Campos MSM, Baratella JRS, Ketzer JC, Mastroti RA, Amarante ACM. Formação e demanda do cirurgião pediátrico no Brasil. Rev Col Bras Cir. [periódico na Internet] 2009; 36(4). Disponível em URL: http://www.scielo.br/rcbc y g 12. Miller GG. Waiting for an operation: parents’ perspectives. Can J Surg. 2004; 17(3):179-81. 13. Carneiro MB, Gouveia VV, coord. O médico e o seu trabalho: aspectos metodológicos e resultados no Brasil. Brasília: CFM; 2004. Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Endereço para correspondência: Lisieux Eyer de Jesus E-mail: lisieux@uol.com.br Endereço para corresp Endereço para corresp Endereço para corresp Endereço para corresp Endereço para corresp Lisieux Eyer de Jesus E-mail: lisieux@uol.com.br 14. Aguiar AS, Jesus LE, Campos MSM, Baratella JRS. Maior popula- ção pediátrica assistida por planos de saúde é o principal y E-mail: lisieux@uol.com.br Rev. Col. Bras. Cir. 2009; 36(4): 356-361
https://openalex.org/W3021758182	https://eprints.kingston.ac.uk/id/eprint/45588/1/Van-der-Heijden-B-45588-VoR.pdf	English	null	A study protocol for a cluster randomised controlled trial on mindfulness-based stress reduction: studying effects of mindfulness-based stress reduction and an additional organisational health intervention on mental health and work-related perceptions of teachers in Dutch secondary vocational schools	Trials	2,020	cc-by	13,832	Janssen et al. Trials (2020) 21:376 https://doi.org/10.1186/s13063-020-4189-3 Janssen et al. Trials (2020) 21:376 https://doi.org/10.1186/s13063-020-4189-3 Open Access A study protocol for a cluster randomised controlled trial on mindfulness-based stress reduction: studying effects of mindfulness- based stress reduction and an additional organisational health intervention on mental health and work-related perceptions of teachers in Dutch secondary vocational schools Math Janssen1* , Yvonne Heerkens1, Beatrice Van der Heijden2,3,4,5,6†, Hubert Korzilius2†, Pascale Peters2,7† and Josephine Engels1† Abstract Background: Dutch teachers in secondary vocational schools suffer from stress and burnout complaints that can cause considerable problems at work. This paper presents a study design that can be used to evaluate the short- term and long-term effectiveness of mindfulness-based stress reduction (MBSR), a person-focused intervention, both within and outside of the context of an additional organisational health intervention. Methods: The proposed study comprises a cluster randomised controlled trial that will be conducted in at least three secondary vocational schools, to which teachers will be recruited from three types of courses: Care, Technology, and Economy. The allocation of the intervention programme to the participating schools will be randomised. The teachers from each school will be assigned to intervention group 1 (IG 1), intervention group 2 (IG 2), or the waiting list group (WG). IG 1 will receive MBSR training and IG 2 will receive MBSR training combined with an additional organisational health intervention. WG, that is the control group, will receive MBSR training one year later. The primary outcome variable of the proposed study is mindfulness, which will be measured using the Dutch version of the Five Facet Mindfulness Questionnaire (FFMQ-NL). In the conceptual model, the effects of teachers’ (Continued on next page) * Correspondence: math.janssen@han.nl †Beatrice Van der Heijden, Hubert Korzilius, Pascale Peters and Josephine Engels contributed equally to this work. 1Occupation and Health Research Group, HAN University of Applied Sciences, Nijmegen, the Netherlands Full list of author information is available at the end of the article * Correspondence: math.janssen@han.nl †Beatrice Van der Heijden, Hubert Korzilius, Pascale Peters and Josephine Engels contributed equally to this work. 1Occupation and Health Research Group, HAN University of Applied Sciences, Nijmegen, the Netherlands Full list of author information is available at the end of the article * Correspondence: math.janssen@han.nl †Beatrice Van der Heijden, Hubert Korzilius, Pascale Peters and Josephine Engels contributed equally to this work. 1Occupation and Health Research Group, HAN University of Applied Sciences, Nijmegen, the Netherlands Full list of author information is available at the end of the article (Continued from previous page) (Continued from previous page) mindfulness resulting from the intervention programmes (MBSR training and MBSR training combined with an additional organisational health intervention) will be related to salient (secondary outcome) variables: mental health outcomes (e.g., burnout, work engagement), work performance, work-related perceptions (job demands and job resources), and personal competencies (e.g., occupational self-efficacy). Data will be collected before (T0) and immediately after the MBSR training (T1), and 3 (T2) and 9 months (T3) after the training. The power analysis revealed a required sample size of 66 teachers (22 in each group). Discussion: The proposed study aims to provide insight into (1) the short-term and long-term effects of MBSR on teachers’ mental health, (2) the possible enhancing effects of the additional organisational health intervention, and (3) the teachers’ experiences with the interventions (working mechanisms, steps in the mindfulness change process). Strengths of this study design are the use of both positive and negative outcomes, the wide range of outcomes, both outcome and process measures, longitudinal data, mixed methods, and an integral approach. Although the proposed study protocol may not address all weaknesses of current studies (e.g., self-selection bias, self-reporting of data, the Hawthorne effect), it is innovative in many ways and can be expected to make important contributions to both the scientific and practical debate on how to beat work-related stress and occupational burnout, and on how to enhance work engagement and work performance. Trial registration: Dutch Trial Register (www.trialregister.nl): NL5581. Registered on 6 July 2016. Trial registration: Dutch Trial Register (www.trialregister.nl): NL5581. Registered on 6 July 2016. K d M df l b d d Add l l h l h M l h l h Keywords: Mindfulness-based stress reduction, Additional organisational health intervention, Mental health, Teachers, Longitudinal approach, Mindfulness, Burnout, Stress, Work engagement, Work performance Background k l d information, and communication sectors, the absenteeism rate in the educational sector is relatively high: 5.3% in the latter versus less than 3% in the former sectors in 2017 [5]. In the Netherlands, the costs of work-related stress absen- teeism for the total workforce is €1.8 billion, of which €275 million involves the costs in the educational sector. Work stress-related absenteeism costs are the highest in the edu- cational sector: almost €6000 (number of days × costs per day) for each employee who is absent [9]. When a teacher is absent, organisations in the educational sector strongly rely on the (mostly serendipitous) availability of substitut- able colleagues to cover for the absent worker. Conse- quently, colleagues are overloaded (i.e., a job demand), while the job resources they can draw from remain the same at best. This pattern creates an imbalance between these colleagues’ job demands and resources, which can jeopardise their well-being [7]. This imbalance between job demands and resources and its associated risk of negative effects on one’s well-being may be an important reason that many novice teachers leave the educational sector within the first 5 years of their career [10] and that many experi- enced teachers retire early. In fact, 45–70% of early retire- ments in the educational sector can be attributed to psychosomatic and psychological problems [7]. Therefore, it is extremely important to reduce and prevent stress and absenteeism in the occupational sector and to develop ef- fective mental health management interventions, which can be both person-focused and organisation-focused. © 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. Page 2 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials (2 Work-related stress and its consequences Mental health interventions in the educational sec- tor are mostly secondary preventive and targeted at the individual level, with the goal of enhancing the ability of teachers to cope with stressors in the workplace [12– 18]. Examples are workshops on stress management skills and mindfulness-based stress reduction (MBSR) programmes. MBSR has been shown to be partly effect- ive in influencing mental health outcomes [19]. leaders/managers. Slutsky et al. [29] conducted a rando- mised controlled trial (RCT) and suggested that small doses of mindfulness training (half-day training) are suf- ficient to increase job productivity, but that larger doses (6-week training) are needed to improve attentional focus at work, job satisfaction, and work-life balance. The systematic review by Donald et al. [30] identified a positive relationship between mindfulness (both opera- tionalised as a personality variable and as an interven- tion) and prosocial behaviour. In a meta-analysis, Klingbeil and Renshaw [31] men- tioned that mindfulness-based interventions with teachers are promising for increasing their mindfulness and psychological well-being and for decreasing psycho- logical distress. Overall, they concluded that their find- ings were similar to the outcomes found in other meta- analyses of the effects of such interventions on em- ployees’ mental health. From a health perspective, primary prevention - when possible - is preferable to secondary and tertiary preven- tion. In their review of occupational stress interventions in Australia, Caulfield et al. [20] suggested that primary interventions generate more positive changes in com- parison with individual-focused secondary or tertiary in- terventions. However, two meta-analyses on work- related stress interventions [21, 22] found no substantial differences between organisational-level and individual- level interventions. One explanation is the complexity of organisational-level interventions, which might hinder the implementation and measurement of outcomes [7, 23]. In view of this, an appropriate (i.e., mixed-methods) evaluation of an organisational health intervention may require consideration of multiple process outcomes to monitor the implementation process and to investigate the outcomes of the intervention in depth [7, 24]. We agree with Van der Klink et al. [25] that there is a need for an integrated approach that combines both an individual-focused intervention and an organisation- focused intervention. Research on mindfulness is often criticised for its poor methodological quality [32, 33]. However, it is impos- sible to conduct such research using a double-blind placebo-controlled design, which is often applied in medical interventions [34]. Work-related stress and its consequences Work-related stress and its consequences Dutch society needs healthy teachers to maintain and im- prove the quality of the education sector and to enhance student performance [1]. In all sectors, however, work- related stress has become an inherent feature of the em- ployment relationship in industrialised countries such as the Netherlands [2]. Work-related stress is an increasingly important cause of workers’ mental health problems, such as stress symptoms, overstrain, and burnout, which can decrease work performance [3, 4]. In 2017, almost one in six Dutch employees reported stress or burnout com- plaints. In the educational sector, this figure was more than one in five employees [5]. More than 30% of teachers have reported that major changes in the work context are an important cause of work-related stress. Teachers are expected to meet higher job demands (e.g., high workload, emotional strain) with fewer job resources, especially less professional autonomy [5]. Job demands can be defined as the physical, social, or organisational aspects of the job that require sustained physical or psychological effort [6]. The increase in teachers’ workload is caused by numerous administrative tasks and school reforms. The growing needs of students also generate emotional strain [7]. Job resources can be defined as the physical, social, or organisational aspects that may help teachers to achieve goals and to stimulate learning and development. As such, job resources can buffer the influence of job demands [6, 8]. Mental health interventions in the educational sector A high percentage of Dutch employees (57%), especially in the educational sector, ask for interventions to Work-related stress is associated with several negative or- ganisational outcomes, such as increased absenteeism and early retirement [5]. In comparison with the agriculture, Janssen et al. Trials (2020) 21:376 Page 3 of 18 Page 3 of 18 Janssen et al. Trials (2020) 21:376 address work-related stress problems [5]. Many em- ployers in this sector (48%) also recognise the risk of stress [5]. Preventive interventions can be classified as primary, secondary, or tertiary. Primary interventions, which are oriented to the organisational level, aim to change the sources of work-related stress. Secondary and tertiary interventions, both of which are focused on the individual employee, aim to decrease stress symp- toms before they cause mental health problems and to treat mental health problems (e.g., burnout), respectively [11]. Individual-focused secondary health intervention: MBSR h h h Individual-focused secondary health intervention: MBSR Two systematic reviews have shown that an MBSR inter- vention programme in the workplace can significantly affect deficit-based outcomes, such as emotional exhaus- tion (one of the three dimensions of occupational burn- out), (occupational) stress, psychological distress, anxiety, and depression [19, 26]. Three systematic re- views also identified significant improvements in asset- based outcomes, such as mindfulness, personal accom- plishment (a dimension of burnout), (occupational) self- compassion, quality of sleep, relaxation, and job per- formance [19, 26, 27]. The systematic review by Donaldson-Feilder et al. [28] reported positive effects on the well-being, resilience, and leadership capability of Work-related stress and its consequences It is obvious that participants cannot be kept blind to the fact that they are (or are not) assigned to an MBSR training programme. This raises questions about which methodological features should be included to improve the research design. Goldberg et al. [32] highlighted six features: (1) active control conditions to consider the amount of non- specific attention participants receive, called the Haw- thorne effect [35]; (2) larger sample sizes; (3) longer follow-up assessment to measure the sustainability of training effects; (4) evaluation of treatment fidelity; (5) reporting of instructors’ skill levels; and (6) intention-to- treat (ITT) analysis. Three other important features are assessing a diversity of outcomes (negative and positive, process and effect measures, mental health and work performance); using a mixed-methods approach that combines quantitative and qualitative data; and combin- ing an individual-focused intervention, such as MBSR, with an additional organisational intervention (i.e., tak- ing an integrated approach) [19]. Aim of the proposed study The proposed study aims to contribute to the debate on prevention of work-related stress and burnout, and improvement of work engagement and work perform- ance by evaluating the short-term and long-term ef- fectiveness of MBSR, as an individual-focused intervention, on teachers in secondary vocational schools, as an example of a possible application area. It will look at the effects of the intervention on teachers’ mental health (mindfulness as the primary outcome), work performance, work-related percep- tions (job demands and job resources), and personal competencies. In addition, it will investigate the ef- fects of a participatory, preventive, organisational health intervention (i.e., a participatory action ap- proach) that targets and engages teachers in a specific course. We hypothesise that participating in the or- ganisational health intervention will positively influence occupational self-efficacy. The application of the organisa- tional health intervention will generate tailored work solu- tions that may positively influence the balance between job demands (work pressure, work-life balance) and job resources (autonomy, feedback, relationships) for all teachers in schools. Figure 1 depicts the conceptual model, including the processes mentioned above and how these relate to mental health outcomes. The right-hand side of the conceptual model presents the study’s secondary outcome variables: mental health outcomes (mindful- ness, burnout, stress, sleep quality complaints, positive and negative emotions at work, work engagement, perceived general health, organisational commitment) and work performance outcomes (work performance and work behaviour, absenteeism) [19]. The relationships between the two interventions (MBSR and MBSR with an additional organisational health intervention), on the one hand, and the two clus- ters of outcome variables, on the other hand, are medi- ated by two clusters of process variables, which are presented in the middle of the model. The first cluster contains personal competencies that represent the per- sonal resource outcomes resulting from the interven- tions (occupational self-efficacy, taking distance, as the opposite of worry) [41–43]. The second cluster contains secondary outcome variables: work-related perceptions that refer to how an individual worker experiences work characteristics. In line with the JD-R model, we make a distinction between job demands (work pressure, emo- tional demands, work-life balance) and job resources (autonomy, feedback from colleagues and superiors, re- lationship with colleagues, relationship with superiors, relationship with students). Organisational health interventions Organisational health interventions The key points of participatory action research (PAR) [36] are the effective ingredients for organisa- tional interventions: having a bottom-up approach; composing a participatory group; fostering active participation by stakeholders (e.g., employees) and collaboration between researchers and stakeholders; using stakeholders’ knowledge, skills, and percep- tions; and creating joint ownership of problems and solutions [7]. Solutions from stakeholders appeared more effective than solutions adopted by others [37]. Page 4 of 18 Page 4 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials (2020) 21:376 The belief that one is the master of one’s own be- haviour and is able to influence others and the en- vironment (i.e., an internal locus of control) is crucial [38]. In other words, the organisational health intervention should target individuals’ occupa- tional self-efficacy: the belief in one’s own ability in a specific domain of work. The most effective way to enhance one’s self-efficacy is through mastery of ex- periences [7, 39]. By taking part in the organisational intervention or even by experiencing its effects, we assume that occupational self-efficacy can be influ- enced to decrease burnout. Indeed, Consiglio et al. [40] found a negative relationship between occupa- tional self-efficacy and burnout, which appeared to be partially mediated by job demands and job resources. job demands and job resources) can influence work stress via two processes. The first process was referred to by Demerouti et al. [6] as the health impairment process, in which high job demands exhaust workers’ mental and physical resources and may therefore lead to a depletion of energy, exhaustion, health problems, and, eventually, premature retirement from their profession. The second process implies a motivational process: job resources have motivational potential that is either in- trinsic (because they foster growth, learning, and devel- opment) or extrinsic (because they are instrumental in achieving work goals) and lead to positive work out- comes [6]. Job resources and personal resources can buf- fer the effects of the job demands [6]. Mindfulness, the primary outcome in our study, can reduce stress by separating work characteristics from employees’ reactions to them. This enables the individ- ual to become aware of the difference between observa- tion and interpretation [46]. Methods/design Study organisation study. They will be asked if they agree to use of their data should they choose to withdraw from the trial. This trial does not involve collecting biological specimens for storage. The proposed study is a cluster randomised controlled trial (CRCT) that uses a mixed-methods design (quanti- tative and qualitative, online questionnaire, telephone and face-to-face interviews) and contains four measure- ment time points (see Fig. 2). The Ethics Committee Practice based Research of het HAN University of Ap- plied Sciences (ECPR) and the Medical Ethics Commit- tee (METC) of Radboud University Medical Centre, both located in Nijmegen, the Netherlands, approved the research proposal (Registration number ACPO 07.12/15; File number CMO: 2019–5266). Both committees stated that the research complied with the requirements of eth- ical conduct of research as set out in the national Code of Conduct for Scientific Integrity in the Netherlands and that it fulfilled the criteria of the Declaration of Helsinki on Ethical Principles for Medical Research In- volving Human Subjects. The study will be carried out in the Netherlands in full compliance with the applicable rules concerning the review of research ethics commit- tees. Participation is voluntary and participants can with- draw at any moment with no consequences. The study title given to the potential participants and other stake- holders is “Mindfulness and job satisfaction of teachers in secondary vocational schools”. Participants will sign informed consent forms before participating in this Significant deviations from the protocol will be docu- mented using a breach report form and will be sent to the funder NWO and to the ethics committees. The protocol in the trial register will be updated. A Standard Protocol Items Recommendations for Interventional Tri- als (SPIRIT 2013) checklist (see Additional file 1) and figure (see Table 1) are provided. Conceptual model For this study, we will use a conceptual model (see Fig. 1) inspired by the job demands-resources (JD-R) model [6, 8] and the literature on mindfulness [19, 41–43]. The JD-R model and the literature on mindfulness present two different but complementary points of view on work stress. The original JD-R model has been expanded to include personal resources, aspects of the self, referring to one’s ability to successfully influence the environ- ment. Examples are self-efficacy, emotional stability, extraversion, and resilience [44, 45]. Both the original and expanded model suggest that job characteristics (i.e., We assume that the five-factor-model of personality, that is the Big Five [47], especially the factors of extra- version and openness, can be expected to positively moderate mental health and work performance. The Big Five consists of five personality characteristics or traits, that are fixed and cannot be developed, in contrast to personal competencies. Page 5 of 18 Janssen et al. Trials (2020) 21:376 (2020) 21:376 Janssen et al. Trials Fig. 1 Conceptual model Participants and recruitment p Study participants will be recruited from the teaching staff at no fewer than three secondary vocational schools. When a secondary vocational school agrees to participate, we will recruit potential participants from three programmes (Care, Technology, and Economy) using e-mail, posters, flyers, and each school’s intranet. The researchers, Human Resources (HR) consultants, and supervisors will inform potential participants about the research project. Respondents who are willing to participate will be screened in terms of the eligibility criteria by the first author (MJ) (see Table 2). Eligible candidates will receive an information letter about the project. This letter in- cludes the information as approved by the ECPR and the Page 6 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials (2020) 21:376 Fig. 2 Flowchart showing the design of the trial study the effects of an additional organisational health intervention that cannot be directed towards selected in- dividuals (i.e., teachers) and to control for “contamin- ation” across individuals (i.e., the effects on one teacher may influence the effects on another teacher in the same course) [49]. In the first secondary vocational school (known as an MBO in Dutch), participating teachers from one course (Care, Technology, or Economy) will be assigned to intervention group 1 (IG 1: MBSR), teachers from another course will be assigned to intervention group 2 (IG 2: MBSR and an additional organisational health intervention), and teachers from the third course METC and the informed consent letter. One week be- fore the start of the interventions, cluster randomisation will be conducted and the participants will be informed about their assignment to one of the intervention groups or the waiting list group. Cluster randomisation A CRCT is a randomised controlled trial in which groups of subjects (i.e., schools) are randomised rather than individual subjects (i.e., teachers) [48]. Cluster ran- domisation will be performed at the school level. This will provide the researchers with the opportunity to Page 7 of 18 Janssen et al. Trials (2020) 21:376 Table 1 SPIRIT checklist Study period Enrolment Allocation Before start Post-allocation TIMEPOINT** t0 Intervention t1 t2 t3 ENROLLMENT: Eligibility screen X Informed consent X [List other procedures] X Allocation X INTERVENTIONS: MBSR training X MBSR training and organizational health intervention X Waiting list group DATA COLLECTION by on line questionnaire: Demographics X Primary outcome and secondary outcomes X X X X Other data variables X X X X DATA COLLECTION by interviews: Expectations X X Experiences X X SPIRIT Standard Protocol Items Recommendations for Interventional Trials, MBSR mindfulness-based stress reduction Interviews were only with some participants in MBSR training and MBSR training and organisational health intervention groups *T0 = before the training; T1 = immediately after the training; T2 = 3 months after the training; T3 = 9 months after the training Table 1 SPIRIT checklist SPIRIT Standard Protocol Items Recommendations for Interventional Trials, MBSR mindfulness-based stress reduction Interviews were only with some participants in MBSR training and MBSR training and organisational health intervention groups *T0 = before the training; T1 = immediately after the training; T2 = 3 months after the training; T3 = 9 months after the training SPIRIT Standard Protocol Items Recommendations for Interventional Trials, MBSR mindfulness-based stress reduction Interviews were only with some participants in MBSR training and MBSR training and organisational health intervention groups *T0 = before the training; T1 = immediately after the training; T2 = 3 months after the training; T3 = 9 months after the training SPIRIT Standard Protocol Items Recommendations for Interventional Trials, MBSR mindfulness-based stress reduction Interviews were only with some participants in MBSR training and MBSR training and organisational health intervention groups *T0 = before the training; T1 = immediately after the training; T2 = 3 months after the training; T3 = 9 months after the training and the project management group, consisting of YH, BVdH, PP and JE, the funder NWO and the ethics com- mittees will be informed. A final decision to terminate the trial will be made by the project management group, deliberating at least every 6 weeks or more, when necessary. Cluster randomisation will be assigned to the waiting list group (WG). The allo- cation will be different at each school (see Table 3). A researcher who is not involved in assigning courses/par- ticipants to the groups will prepare concealed, consecu- tively numbered, sealed opaque envelopes. Every envelope will contain a paper indicating the treatment assignment at school level (type 1, 2 or 3). The MBO schools will receive their envelopes from a researcher who is unaware of the randomisation sequence. The MBO schools can open the envelope in the presence of the researcher and the researcher will be informed about the treatment assignment. The trial conduct will be audited by an annual evalu- ation report for the funding organisation NWO. The re- port is also available for the ethics committees and the project management group, which will discuss the pro- gress of the trial every 6 weeks. YH and JE will be re- sponsible for the daily supervision of the trial. The implementation of the interventions and the data collec- tion will be strictly separated. There is little evidence of harmful effects of MBSR [19]. Also, we will recruit participants from a healthy target population, therefore, there will be no special cri- teria for discontinuing or modifying the allocated inter- ventions. In the case of (serious) adverse events and harms from the intervention, the participant concerned will be referred to an occupational health professional Procedures All participants will receive the other three follow-up ques- tionnaires on a secured website after the MBSR training (T1), 3 months later (T2), and 9 months after the MBSR training (T3). The first author will conduct face-to-face interviews with at least 12 participants from IG 1 and 12 participants from IG 2 at T1 and T3. At T1, some mem- bers of the participatory group that will be involved in the organisational health intervention - excluding the teachers participating in IG 2 (e.g., a superior, an HR consultant, and the director of the programme) - will be interviewed about the process and effects of the organ- isational health intervention. different for each school) (T0). After completing the questionnaire, the participating schools will be randomly assigned (type 1, 2, or 3; see Table 3), meaning that par- ticipants will take part in IG 1, IG 2, or WG depending on the course where they are working. At T0, the first author will conduct 10-min telephone interviews with at least 12 participants from IG 1 and 12 participants from IG 2 about their expectations of the interventions. All participants will receive the other three follow-up ques- tionnaires on a secured website after the MBSR training (T1), 3 months later (T2), and 9 months after the MBSR training (T3). The first author will conduct face-to-face interviews with at least 12 participants from IG 1 and 12 participants from IG 2 at T1 and T3. At T1, some mem- bers of the participatory group that will be involved in the organisational health intervention - excluding the teachers participating in IG 2 (e.g., a superior, an HR consultant, and the director of the programme) - will be interviewed about the process and effects of the organ- isational health intervention. Additional organisational health intervention The organisational intervention that will be used in the proposed study will be developed following a design- based approach [55] in accordance with the key points of PAR [36] with a grounding in the JD-R model [6]. A design-based approach is pragmatic, based on theory, observations and experiences [56]. The organisational intervention will be developed following the steps of the design-based approach (see Fig. 3). The JD-R model [6, 45] assumes a relationship between work characteristics (i.e., job demands and job resources) and work out- comes. High job-demands lead to stress reactions and unhealthiness (exhaustion process), while high job- resources increase motivation and productivity (motiv- ational process). Participants in IG 1 and IG 2 should attend at least four of the nine MBSR sessions, because Bear et al. [50] revealed that structural changes in perceived stress did not occur until after four MBSR sessions [19]. Partici- pants in WG will attend a MBSR programme one year later. The organisational health intervention will consist of two phases. In the first phase, the “needs assessment phase”, we will start with assessment of needs for the implementation of work-oriented solutions, using the knowledge, skills, and perceptions of teachers and edu- cational managers to investigate the positive points (job resources) and the main difficulties (job demands) in the specific course. In the second phase, the “implementa- tion phase”, the teachers and managers will jointly deter- mine the highest priorities and develop a feasible work- related action/implementation plan. The collected data will be stored on a secure disk to ensure confidentiality. Not the researcher (MJ) but an in- dependent external organisation, assigning encrypted numbers to the participants, will collect the data. The researcher (MJ) cannot link the numbers and the partici- pants. Only the researcher (MJ), the members of the project management group and a methodologist (HK) will have access to the data. A participatory group will be formed in the needs as- sessment phase. It will preferably include two teachers participating in the MBSR training, two other teachers (workplace), the HR consultant for the specific course (ex- pert), a supervisor, the course director (decision-making power), an external facilitator, and relevant others from the workplace (e.g., a union member or a member of the formal employee participation committee). Procedures All study participants will be asked to complete an on- line questionnaire on a secured website before the start of the intervention(s) (the starting date of the study is Table 2 Eligibility criteria Inclusion criteria Exclusion criteria Teachers in the Care, Technology, and Economy courses Attended mindfulness training in the past 2 years Employed in a secondary vocational school for at least 2.5 days a week for at least 1 year Attended stress reduction training (e.g., cognitive therapy or relaxation training) in the past 2 years Page 8 of 18 Page 8 of 18 Janssen et al. Trials (2020) 21:376 Table 3 Cluster randomisation Care Technology Economy MBO school, type 1 IG 1a IG 2b WGc MBO school, type 2 WG IG 1 IG 2 MBO school, type 3 IG 2 WG IG 1 a IG 1: intervention group 1 (MBSR) b IG 2: intervention group 2 (MBSR and an additional organisational health intervention) c WG: waiting list group (control group that will receive MBSR one year later) particular, a 3-min breathing space and psycho- education about the nature of thoughts. The MBSR programme will consist of eight 2.5-h weekly group ses- sions, each with 4–15 participants per group, homework involving 45 min of daily home exercise 6 days a week, and one day with 7-h of silence. The sessions will be su- pervised by one of the four recruited, qualified, mindful- ness trainers, who will receive a training script. The first session will begin with a short introduction to the programme and meet and greet between participants. Each session will consist of different meditation exer- cises, enquiry, psycho-education, and a specific theme (see Table 4). At the end of each session, participants will be given homework that will be discussed in the subsequent session. different for each school) (T0). After completing the questionnaire, the participating schools will be randomly assigned (type 1, 2, or 3; see Table 3), meaning that par- ticipants will take part in IG 1, IG 2, or WG depending on the course where they are working. At T0, the first author will conduct 10-min telephone interviews with at least 12 participants from IG 1 and 12 participants from IG 2 about their expectations of the interventions. MBSR: main intervention MBSR, developed by Kabat-Zinn [51], is the most com- mon form of secular mindfulness-based training [52]. MBSR aims to reduce suffering or stress [53] and was originally developed for patients with chronic pain. This training programme is primarily based on Kabat-Zinn’s curriculum [51], but it contains elements of mindfulness-based cognitive therapy (MBCT) [54]: in The focus of the intervention is to stimulate dialogue between management and employees/teachers in which they can jointly investigate improvement opportunities and implement solutions that reduce stress and improve Page 9 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. MBSR: main intervention Trials Table 4 Content of MBSR group sessions Session Theme Content of group sessions Homework 1 Automatic pilot • Introduction • Raisin-eating exercise • Body scan • Body scan • Attention to routine activity • Eating one meal mindfully 2 Perceiving clearly • Body scan • Imagery exercise to demonstrate the relationship between thoughts and feelings • Sitting meditation, paying attention to breathing • Body scan • Attention to breath • Awareness of pleasant events • Attention to routine activity 3 From doing to being: a mode of doing and a mode of being • Lying-down yoga exercises • Sitting meditation with a focus on breathing, bodily sensations, sounds • Pleasant events • Seeing exercise to demonstrate the difference between observation and interpretation • Three-minute breathing space (mini-meditation) • Body scan • Lying-down yoga exercises • Attention to breath • Awareness of unpleasant events 4 Be present • Three-minute breathing space (mini-meditation) • Standing yoga exercises • Unpleasant events; interrelatedness of bodily sensations, feelings, and thoughts • Sitting meditation with a focus on breathing, bodily sensations, sounds, feelings/ emotions, and thoughts • Body scan • Standing yoga exercises • Sitting meditation • Three-minute breathing space • Awareness of stress reactions 5 Recognising and allowing what really is: reacting versus responding • Three-minute breathing space (mini-meditation) • Walking meditation • Sitting meditation with a focus on breathing, bodily sensations, sounds, feelings/ emotions, thoughts, and random attention • Automatic stress reactions and stress response • Mid-term evaluation • Meditation by choice • Three-minute breathing space • Awareness of difficult situations • Awareness of reactions in difficult situations 6 Mindful communication • Standing yoga exercises • Sitting meditation with a focus on breathing, bodily sensations, sounds, feelings/ emotions, thoughts, and random attention • Mindful communication exercises • Meditation by choice • Three-minute breathing space Day of silence Deepen mindfulness skills in silence • Various meditation exercises • Silent lunch and tea break 7 Taking care of yourself: balance in life • Standing/lying yoga exercises • Sitting meditation • Communication exercises • Meditation exercises without CD • Attention to routine activities 8 The rest of your life • Different exercises • Own menu of mindfulness exercises • Maintaining practice: review of supports • Reflection on training • Saying goodbye • Further sources of information MBSR Mindfulness-based stress reduction • Meditation exercises without CD • Attention to routine activities • Further sources of information • Reflection on training MBSR Mindfulness-based stress reduction MBSR Mindfulness-based stress reduction participatory group that includes teachers, the HR consultant, the director, and the managers will enable the group to establish the highest priorities and develop solutions to improve the working environment. MBSR: main intervention participatory group that includes teachers, the HR consultant, the director, and the managers will enable the group to establish the highest priorities and develop solutions to improve the working environment. work pleasure. The intervention consists of approxi- mately five sessions (see Table 5), starting with assess- ment of needs to the implementation of work-oriented solutions. The programme theory or critical assumptions under- pinning the organisational health intervention (How would the intervention work if it were successfully imple- mented?) [57] are as follows: 2. A supported, basic work-related action plan will be developed as well, consisting of: “relatively easy and quick to solve” priorities (= quick wins), solutions, a timeline, necessary resources, and an implementa- tion method. 1. Involving an external facilitator (an expert in organisational change processes) and creating a 3. The quick wins will be implemented immediately. Page 10 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials Fig. 3 Steps of the design-based approach Fig. 3 Steps of the design-based approach Baseline characteristics of participants at T0 Baseline characteristics of participants at T0 4. Healthy working in the course will be improved, dialogue between management and employees/ teachers will be stimulated, and occupational self- efficacy will be increased. 4. Healthy working in the course will be improved, dialogue between management and employees/ teachers will be stimulated, and occupational self- efficacy will be increased. Gender Age (years) Family situation Level of education Nature of employment; course (Care, Technology, or Economy) Years of work experience Number of working days and hours Waiting list group The participants on the waiting list will receive MBSR after one year. They cannot attend a mindfulness train- ing course or stress reduction training (e.g., cognitive therapy or relaxation training) until T3. Nature of employment; course (Care, Technology, or Economy) Years of work experience Number of working days and hours Secondary outcomes Secondary mental health outcomes Burnout will be measured using the Dutch version of the Maslach Burn- out Inventory - Education Survey (MBI-ES), the Utrechtse BurnOut Schaal-Leerkrachten (UBOS-L; Ut- recht Burnout Scale - Education) [60–62]. The 22-item UBOS-L has a three-dimensional structure with the fol- lowing subscales: emotional exhaustion, mental distance (cynicism, depersonalisation), and (job-related) personal accomplishment/professional efficacy. The total scores of the three subscales range from 0 to 6. Higher values indicate more emotional exhaustion, more mental dis- tance, and more personal accomplishment, respectively. Maslach et al. [61] reported Cronbach’s alpha for the three subscales - emotional exhaustion (8 items), mental distance (7 items), and professional efficacy (7 items) - of 0.91, 0.73, and 0.85, respectively. The emotional ex- haustion subscale is highly correlated with other mental and physical complaints, and with job demands like time pressure [61]. Mental distance and professional efficacy are significantly related to personal resources like auton- omy and ambition level [61]. Perceived general health will be measured using two items (1 and 11) from the Dutch version of the Short Form 36 Health Survey, version 2 (SF-36-v2), named RAND-36 [69]. The score on each item ranges from 1 to 5; the transformed overall score on the two items varies from 0 to 100. Higher values indicate higher levels of perceived general health. The internal consistency (Cronbach’s alpha) as reported by van der Zee et al. [69] is 0.81. Organisational commitment will be assessed by four items derived from the four-item Affective Commitment Scale (ACS) used by Smeek et al. [70], who reported re- ported Cronbach’s alpha of 0.70 for this scale. Stress will be assessed using the 14-item stress scale of the Dutch 42-item Depression, Anxiety, Stress Scales (DASS) [63]. The total score on the stress scale ranges from 0 to 21. Higher values indicate more stress. The DASS has a three-factor structure: depression, anxiety, and stress. Nieuwenhuijsen et al. [63] reported internal consistency of the DASS of 0.94, 0.88, and 0.93, respectively. Secondary work performance outcomes Work per- formance and work behaviour, defined as behaviours or actions of employees that are relevant to the organisa- tion’s goals, will be measured using the Dutch Indivi- duele WerkPrestatie Vragenlijst (IWPQ; Individual Work Performance Questionnaire) [71]. The 18-item questionnaire consists of three subscales: task perform- ance (5 items), contextual performance (8 items), and counter-productive work behaviour (5 items). Baseline characteristics of participants Measures of the baseline characteristics of the partici- pants are gender, age (years), family situation, level of education, nature of employment (course Care, Technol- ogy, or Economy), years of work experience, and number of working days and hours. Mindfulness skills will be examined using the Dutch ver- sion of the Five Facet Mindfulness Questionnaire (FFMQ- NL), a self-report measure based on a factor analysis of items from the five most widely used mindfulness question- naires [58, 59]. The 39-item FFMQ-NL has a five-factor structure that is captured in the following five subscales: observing, describing, acting with awareness, non-judging of inner experience, and non-reactivity to inner experience. The FFMQ-NL total score ranges from 39 to 195; the total scores of the subscales are 8–40, except for non-reactivity (7–35). Higher values indicate higher levels of mindfulness skills. De Bruin et al. [59] reported internal consistency of 0.85 (Cronbach’s alpha) for the FFMQ-NL total score (for the non-meditating sample) and 0.90 (for the meditating sample); Cronbach’s alpha for the five subscales varies from 0.70 to 0.89 [59]. There is modest but significant correlation between the five dimensions (ranging from 0.13 to 0.39), which suggests that they represent distinct but interrelated constructs [59]. All mindfulness dimensions are positively correlated with meditation experience and negatively corre- lated with psychological symptoms (i.e., depression, anxiety, insomnia, and social withdrawal) [59]. All the dimensions except for observing are negatively related to the constructs Table 5 Sessions and content of participatory group sessions Session Content of participatory group sessions Phase 1 • Introduction • Mutual expectations, drive and mission/vision. Mutual commitment • How to engage colleagues? • First inventory of positive points (job resources) and difficulties (job demands) for work pleasure in the course, as inspired by the JD-R model (Prioritising based on importance, level of influence, and the wait time for results) 1 2 • Which priorities do we choose? • Possible solutions • Action plan • Implementation plan 1 3 • Follow up 2 4 • Follow up 2 5 • Follow-up implementation • Maintaining, continuation, evaluation • Saying goodbye to the external facilitator 2 Page 11 of 18 Page 11 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials of alexithymia (lack of ability to identify and describe feel- ings, and lack of interest in feelings, cognitions, and motiva- tions), thought suppression, rumination, worry and dissociation [59]. Baseline characteristics of participants Overall, the psychometric properties of the FFMQ-NL [60] are comparable to those of the original English version [58]. values indicate more positive emotions and more nega- tive emotions, respectively. The positive emotions sub- scale is negatively correlated with the frequency (r = − 0.22) and duration (r = −0.23) of future absenteeism of managers; the negative emotions subscale is not corre- lated with these variables [66]. Work engagement will be assessed using the nine-item Dutch version of the shortened Utrecht Work Engage- ment Scale (UWES), the UBES-9 [67, 68]. The three- dimensional UWES consists of three 3-item subscales: vigour, dedication, and absorption. The total score on the UWES ranges from 9 to 54. Higher values indicate more work engagement. Schaufeli et al. [67] reported in- ternal consistency (Cronbach’s alpha) for the total UBES-9 of 0.93 and the alpha for the three subscales varies from 0.79 to 0.89. The three work engagement scales are highly correlated (minimum r = 0.65) [67]. The three factors are negatively correlated with the three di- mensions of burnout [67]. Secondary outcomes The total scores on the three subscales range from 0 to 4. Higher values indicate more task performance, more contextual performance, and more counterproductive work behav- iour. The internal consistency (Cronbach’s alpha), re- ported by Koopmans et al. [71], varies between 0.78 (task performance) and 0.85 (contextual performance). Task performance and contextual performance are mod- erately positively correlated with work engagement: r values 0.32 and 0.43, respectively. Counterproductive work behaviour is moderately negatively correlated with work engagement (r value −0.29) [71]. Sleep quality complaints will be measured using the Dutch sleep quality subscale of the 14-item Vragenlijst Beleving en Beoordeling van de Arbeid 2.0 (VBBA2.0; Perception and Assessment of Labour 2.0 Question- naire). The total score ranges from 0 to 100. Higher values indicate more complaints and lower-quality sleep. Van Veldhoven et al. [64] reported internal consistency (Cronbach’s alpha) of 0.90. Positive and negative emotions at work will be assessed by the 12-item Dutch version of the Job-related Affective Well-Being Scale (JAWS) [65, 66]. The Dutch JAWS has a two-factor structure, which is reflected in the following two subscales: a positive six-item emotions scale (Cronbach’s alpha = 0.77) and a negative six-item emotions scale (Cronbach’s alpha = 0.78) [66]. The total score on each subscale varies from 6 to 30. Higher Page 12 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials (2020) 21:376 Absenteeism, working fewer than the normal hours or days in the employment contract due to a health prob- lem, will be measured by four items from the NEA 2018 [72], the Dutch Working Conditions Survey 2018 (e.g., How many working days have you been absent in the last three months? How many times have you been absent in the last 12 months over one or more periods longer than 2 weeks? If so, has the absenteeism to do with your work? Have you fully returned to work now?). internal consistency (Cronbach’s alpha) of the negative HWI and the negative WHI, as reported by Geurts et al., is 0.72 and 0.85, respectively [74]. The job resources of autonomy (4 items), feedback from colleagues and superior (4 items), relationship with colleagues (6 items), relationship with superior (6 items), and relationship with students (4 items) will be mea- sured using several scales of the VBBA2.0. The total score on every scale ranges from 0 to 100. Process evaluation of the MBSR training A process evaluation will be conducted to explore work- ing mechanisms and possible barriers to MBSR in this population. The process evaluation of MBSR will be conducted using both quantitative (online questionnaire, primarily questions about experiences with the MBSR training) and qualitative measurements (semi-structured interviews). All participants will receive the online ques- tionnaire at T0, T1, T2, and T3. A selection of the partici- pants in IG 1 and IG 2 will be interviewed at T0, T1, and T3. The interview at T0, lasting 10 min, will be con- ducted by telephone and will be focused on expectations about MBSR. The face-to-face interview at T1, lasting 25– 35 min, will be about experiences during the MBSR training and its short-term effects. The face-to-face interview at T3, lasting approximately 25–35 min, will be focused on long-term effects. All interviews will be re- corded, fully transcribed, and anonymised. A deductive qualitative analysis will be performed, because of the availability of a focused main research question and a conceptual model [75, 76]. The interviews can provide valuable information about the working mechanisms and possible barriers of the MBSR training. “Work-related perceptions” The job demands of work pressure and emotional demands will be measured using the six-item Werktempo & Werkhoeveelheid (Work Pace and Workload) questionnaire and the five-item Emotionele Belasting (Emotional Demands) question- naire of the VBBA 2.0, respectively. The total score ranges from 0 to 100. Higher values indicate more work pressure and more emotional demands. Van Veldhoven et al. [64] reported an internal consistency (Cronbach’s alpha) of the subscales of 0.86 and 0.80, respectively. The job demand of balance between work and private life will be assessed using two subscales of the Dutch version of the Survey Work-home Interaction-NijmeGen (SWING): the negative Work-Home Interaction (nega- tive WHI) subscale, which measures negative effects of work on functioning at home and the negative Home- Work interaction (negative HWI) subscale, which mea- sures negative effects of home on functioning at work [74]. The SWING also includes two other subscales: the positive Work-Home Interaction (positive WHI) sub- scale and the positive Home-Work interaction (positive HWI) subscale. The total score on the negative WHI and the negative HWI ranges from 0 to 3. Higher values indicate more problems in work-home interaction. The Secondary outcomes Higher values indicate more problems in the specific outcomes (e.g., a higher score on autonomy indicates less autonomy). The internal consistency (Cronbach’s alpha) of the subscales, reported by van Veldhoven et al. [64], varies from 0.81 to 0.87. Moderating variable “personality characteristics” Moderating variable “personality characteristics” Moderating variable personality characteristics The Dutch version of the Ten Item Personality Inven- tory (TIPI) will be used to measure the dimensions of the five-factor-model of personality: neuroticism, extra- version, openness, agreeableness, and conscientiousness. Each factor will be assessed by two unipolar items with a 7-point Likert scale ranging from 1 = not applicable at all to 7 = completely applicable. The TIPI has been shown to be a valid alternative for the existing extensive Big Five instruments [47]. Taking distance, which comprises not worrying or ru- minating about work at home, will be assessed using the three-item “Afstand Nem”’ (Taking Distance) subscale of the VBBA 2.0. The total score varies from 0 to 100. Higher values indicate that the individual experiences more problems with taking a distance from work. Van Veldhoven et al. [64] reported internal consistency (Cronbach’s alpha) of the subscale of 0.80. Mediating variables “Personal competencies outcomes” or “process- focused outcome measures” Occupational self-efficacy, which refers to the confidence a worker has in their per- ceived ability to perform job tasks successfully, will be assessed using the short (six-item) Dutch version of the Occupational Self-Efficacy Scale [73]. The total mean score ranges from 1 to 6. High values reflect high occu- pational self-efficacy. Rigotti et al. [73] reported internal consistency (Cronbach’s alpha) of 0.85. Process evaluation of the additional organisational health intervention A process evaluation of the additional organisational health intervention will be performed to assess the Page 13 of 18 Page 13 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials (2020) 21:376 variables, and as frequencies and percentages for cat- egorical variables. The outcomes of the questionnaires will be compared at baseline (T0), immediately after the intervention(s) (T1), 3 months later (T2), and 9 months after the intervention(s) (T3). All analyses will be con- ducted according to the intention-to-treat (ITT) principle. ITT analysis, based on the initial treatment al- location and not on the treatment eventually received, will avoid the effects of drop-out, and as such we pre- vent breaking the random allocation to the intervention groups [79]. Per-protocol (PP) analyses in the treatment- adherent sample (i.e., participants in IG 1 and IG 2 have to attend at least four of the nine MBSR sessions, and participants in WG cannot attend a MBSR programme or stress reduction training) will also be performed. The aim of PP analysis is to assess the effects of MBSR and the additional organisational health intervention under optimal conditions: what is the effect if participants are fully compliant [80]? Therefore, drop-outs need to be excluded from any PP analysis. requirements/conditions for successful implementation, based on a simplified version of the theoretical frame- work presented by Nielsen and Randall [57, 77]. These researchers indicate that a process evaluation is import- ant because the implementation process can moderate or mediate the potential effects of the intervention on health and well-being [57, 77]. Successful implementa- tion is a prerequisite for exposure to the intervention and therefore for entailing possible health effects. The framework, which enables us to link intervention pro- cesses to intervention outcomes, will be applied to quali- tatively appraise 3 themes of process components: (1) intervention design and implementation, determining the maximum level of intervention exposure; (2) inter- vention context; and (3) participants’ mental models [57, 77]. The process components of themes 2 and 3 may mediate or moderate the link between any intervention exposure and intervention effects [77]. Table 6 lists the themes and requirements/process components for suc- cessful implementation that will be assessed in the semi- structured interviews (T1). Applying the framework will help us to understand why the implementation process was successful or not [57]. Blinding Participants, trainers, facilitator, and researchers cannot be blinded to their assigned intervention after cluster randomisation. All participants have to fill in the online questionnaire at home or at work, excluding the influ- ence of the researcher. The developer of the online ques- tionnaire will collect the data and provide the anonymous data to the researcher. The researcher will analyse the data blinded to the assigned intervention. All statistical analyses will be conducted using IBM SPSS Statistics, version 25. The level of significance will be set at 0.05. The analysis of the qualitative data, col- lected by the semi-structured interviews at T0, T1, and T3, will be deductive [−76], and will be conducted by means of using ATLAS.ti [83]. Process evaluation of the additional organisational health intervention The quantitative short-term and long-term effects of MBSR and the additional organisational health intervention (differences between T0, T1, T2, and T3) will be examined using longitudinal regression analysis (generalised estimat- ing equations, GEE, or mixed models), which is fit to ana- lyse longitudinal/clustered data in clinical trials [81] or repeated-measures designs (general linear model, GLM) [82]. The baseline values of outcomes (T0) of the three groups (IG 1; IG 2; WG) will be defined as independent variables, while the outcomes on the follow-up measure- ments (T1, T2, T3) will be treated as dependent variables. Correction of confounding variables will be applied. The process evaluation will be conducted using semi- structured interviews. A selection of participants in IG 2 (taking into account participating in the participatory group or not) and of other participatory group members who are not participating in the MBSR training (e.g., teachers not participating in the MBSR training, or supervisor, director, HR consultant, work council member, trade union mem- ber) will be interviewed at T0, T1, and T3. To investigate the working mechanisms (How is men- tal health improved?) of MBSR and the organisational health intervention, mediating and moderating analyses will be conducted. The mediating effect of personal competencies on mental health outcomes and on work performance outcomes will be tested. The mediating ef- fect of work-related perceptions on mental health out- comes and on work performance outcomes will also be investigated. The moderating effect of the Big Five, espe- cially as regards the factors of extraversion and open- ness, on mental health outcomes/work performance outcomes will also be examined. Sample size A power analysis (GPower; version 3.9.1.4) revealed that a sample size of 22 participants in each group (IG 1; IG 2; WG), with at least two repeated measurements would enable detection of a medium effect size (d = 0.50) [78], with power of 0.95 and alpha of 0.05. A total sample size of 66 participants is therefore required. Statistical analyses Baseline characteristics of participants will be presented as means and standard deviations (SDs) for metric Results of the trial will be communicated by scientific articles in open access journals, management letters for Page 14 of 18 Janssen et al. Trials (2020) 21:376 Table 6 Themes and requirements/process components for successful implementation, based on a simplified version of the theoretical framework from Nielsen and Randall [77] Themes and requirements Operationalisation 1) Intervention design and implementation Initiation Commitment to the intervention and the motivation of the director and team managers Communication about the intervention at the start Communication to the teachers from the course, the mindfulness training participants, and the participatory group members Participation • Establishment of a participatory group • Involvement of the teachers in the course and of the participants in the mindfulness training and in the participatory group Targeting Choosing the right problems in the workplace with the possibility of quick wins Satisfaction The teachers’/participants’ satisfaction with the intervention 2) Intervention context Organisation’s culture Inherent features of the organisation’s culture that facilitate or impede the implementation of the action plan Conditions The organisation’s capacity and skills to implement the action plan Events Events that interfere with implementation of the action plan 3) Participants’ mental models Readiness to change Employees’ and participants’ readiness to change at T1 Perceptions Was the perception of the intervention (action plan) positive? T1 timepoint 1 (immediately after mindfulnesss-based stress reduction training) Table 6 Themes and requirements/process components for successful implementation, based on a simplified version of the theoretical framework from Nielsen and Randall [77] T1 timepoint 1 (immediately after mindfulnesss-based stress reduct emotions at work, work engagement, organisational commitment), but also work performance and work- related perceptions (e.g., job demands and job re- sources). Process measures (e.g., occupational self- efficacy, taking distance) will also be examined. Hence a wide range of outcomes will be measured [19]. participants and non-participants of Dutch secondary vocational schools, and articles for professional maga- zines intended for occupational health professionals. Availability of data and materials Other than the authors no other entities have contractual agreements with regard to access to the final dataset. g The datasets generated and/or analysed during the current study are not publicly available due to the ongoing research, but are available from the corresponding author on reasonable request. Discussion More specific- ally, it considers not only mental health (e.g., positive Page 15 of 18 Page 15 of 18 Janssen et al. Trials (2020) 21:376 characteristics of the teacher and the environment (work and personal circumstances) [6, 84]. Funding This study is funded by a grant of NWO, Dutch Organization for Scientific Research (file number: 023.007.051, www.nwo.nl. MJ received the funding). The funder has/had no role in the design, data collection, analysis, or interpretation of the data; the writing of the manuscript; or the decision to submit the study protocol for publication. Another potential source of bias is associated with the effect of attention received by teachers in IG 1 and IG 2, also known as the Hawthorne effect [35]. Participating in group sessions in IG 1 and IG 2 may lead to an over- estimation of the effect of MBSR and the organisational health intervention. Participants cannot be blinded to the allocated intervention, so the Hawthorne effect can- not be excluded. Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s13063-020-4189-3. Despite the many strengths of the proposed study, complying with research ethics implies that we cannot account for some limitations that have already been raised in previous literature. For example, the proposed study design will be based on self-selection, as we de- pend on voluntary participation by teachers, which may result in somewhat biased samples. Moreover, it is likely that the characteristics of the teachers who participate in the proposed study (e.g., motivation, sensitivity to the MBSR training and the organisational health interven- tion, personality) may differ from those who will not participate or those who drop out early. Additional file 1. SPIRIT 2013 Checklist: Recommended items to address in a clinical trial protocol and related documents. Trial status The trial is funded for 5 years. The first participants were randomly assigned in September 2016. Final out- come assessments will be completed in June 2020. This is the first protocol version (31 August 2015). The design of the cluster randomised controlled trial (CRCT), in which schools are randomised as opposed to individual teachers, is another strength of our proposed approach. CRCT allows us to study the effects of an or- ganisational health intervention and to control for “con- tamination” across participants [48, 49]. Authors’ contributions JE MJ d YH d JE, MJ, and YH acquired the funding. BvdH, JE, MJ, PP, and YH designed the study. MJ and YH are responsible for the acquisition of data. BvdH, HK, JE, MJ, PP, and YH are responsible for the analysis and interpretation of data. MJ drafted the manuscript. All authors were involved in revising the manuscript. All authors read and approved the final version of the manuscript. All named authors adhere to the authorship guidelines of Trials. All authors have agreed to publication. Data from questionnaires using self-reports may be biased [85]. The primatologist and psychologist De Waal [86] posited that human beings are insufficiently aware of their inner state and may therefore mislead them- selves and others. The study of the human psyche needs behavioural reports, based on observation by others [86]. The proposed study will address this concern partly by using both validated questionnaires and data triangula- tion (both quantitative and qualitative data). Abbreviations ACS: Affective Commitment Scale; CRCT: Cluster randomised controlled trial; DASS: Depression, Anxiety, Stress Scales; ECPR: Ethics Committee Practice- based Research of het HAN University of Applied Sciences, Nijmegen; FFMQ- NL: Five Facet Mindfulness Questionnaire; GEE: Generalised estimating equations; GLM: General linear model; HR: Human resources; HWI: Home- work interaction; IG 1: Intervention group 1; IG 2: Intervention group 2; ITT: Intention-to-treat analysis; IWPQ: Individual Work Performance Questionnaire; JAWS: Job-related Affective Well-being Scale; JDR: Job demands-resources; MBCT: Mindfulness-based cognitive therapy; MBI- ES: Maslach Burnout Inventory - Education Survey; MBSR: Mindfulness-based stress reduction; METC: Medical Ethics Committee; METC: Medical Ethics Committee of Radboud University Medical Centre, Nijmegen; PAR: Participatory action approach; PP: Per-protocol analysis; SDs: Standard deviations; SF-36v2: Short Form 36 Health Survey, version 2; SWING: Survey Work-home Interaction-NijmeGen; TIPI: Ten Item Personality Inventory; UBOS- L: Utrechtse BurnOut Schaal-Leerkrachten (Utrecht Burnout Scale - Educa- tion); UWES: Utrecht Work Engagement Scale (Dutch; UBES); VBBA2.0: Vragenlijst Beleving en Beoordeling van de Arbeid 2.0 (Perception and Assessment of Labour 2.0); WG: Waiting list group; WHI: Work-home interaction ACS: Affective Commitment Scale; CRCT: Cluster randomised controlled trial; DASS: Depression, Anxiety, Stress Scales; ECPR: Ethics Committee Practice- based Research of het HAN University of Applied Sciences, Nijmegen; FFMQ- NL: Five Facet Mindfulness Questionnaire; GEE: Generalised estimating equations; GLM: General linear model; HR: Human resources; HWI: Home- work interaction; IG 1: Intervention group 1; IG 2: Intervention group 2; ITT: Intention-to-treat analysis; IWPQ: Individual Work Performance Questionnaire; JAWS: Job-related Affective Well-being Scale; JDR: Job demands-resources; MBCT: Mindfulness-based cognitive therapy; MBI- ES: Maslach Burnout Inventory - Education Survey; MBSR: Mindfulness-based stress reduction; METC: Medical Ethics Committee; METC: Medical Ethics Committee of Radboud University Medical Centre, Nijmegen; We assume that many participants experience work pressure, time pressure, and stress complaints. The MBSR training (and the additional organisational partici- patory health intervention) requires a lot of time and ef- fort from the teachers. We are therefore aware that (potential) participants have to be motivated to prevent premature drop-out. In line with this, the time frame of the organisational health intervention (8–12 weeks) is a short period with regards to capturing organisational changes. Therefore, the T1 measurement might occur too soon to detect ef- fects. However, the measurements at T2 and T3 over- come this limitation. Discussion The proposed study will evaluate the short-term (T1) and long-term effects (T2 and T3) of mindfulness-based stress reduction (MBSR), a person-focused intervention aimed at strengthening the individual capacity of teachers in sec- ondary vocational schools to cope with stress and enhance their mental health. In addition, the possible enhancing ef- fects of an additional organisational health intervention, a participatory action approach, will be investigated as well. The teachers’ experiences with the interventions (the working mechanisms of MBSR and the organisational health intervention) will also be examined. This study is a cluster randomised controlled trial, in which intervention group 1 (IG 1; receiving MBSR) and intervention group 2 (IG 2; receiving MBSR and an additional organisational health intervention) will be compared with the waiting list group (WG; the control group). In their systematic review on MBSR and employees’ mental health, Janssen et al. [19] reported that 14 of the 23 studies included in the review only incorporated short-term effects, measured immediately after the inter- vention. However, in terms of the cost-benefit ratio, MBSR and the organisational health intervention should lead to sustainable long-term effects. Therefore, another strength of this study is that it will gather longitudinal data by measuring both short-term and long-term effects (until 9 months after the intervention). The proposed study will use a mixed-methods ap- proach, which is rare in studies on the effects of MBSR [19]. That approach means that, in addition to quantita- tive data, qualitative data will be collected to investigate in-depth relevant process measures and to capture the mechanisms by which MBSR (key aspects of the MBSR programme) and the organisational health intervention (factors for successful implementation in an organisa- tion) result in specific outcomes. Another strength of the proposed study is the integrated approach, which combines an individual-focused secondary intervention (MBSR) and an organisation-based primary intervention. This is important since teachers’ stress likely results from a complex interaction between personal Many previous studies on the effects of MBSR on em- ployees have primarily assessed negative outcomes, fo- cusing predominantly on mental health (e.g., burnout, stress level, psychological distress). Process measures, which are suitable for investigating how mindfulness can contribute to well-being, have rarely been assessed [19]. The strength of this study lies in the fact that we will as- sess both negative and positive outcomes. References 18. Taylor C, Harrison J, Haimovitz K, Oberle E, Thomson K, Schonert-Reichl K, Roeser W. Examining ways that a mindfulness-based intervention reduces stress in public school teachers: a mixed-methods study. Mindfulness. 2016; 7(1):115–29. https://doi.org/10.1007/s12671-015-0425-4. 1. Van der Steeg M, Gerritsen S. Teacher evaluations and pupil achievement: evidence from classroom observations [CPB Discussion Paper 230]. 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Author details 1 1Occupation and Health Research Group, HAN University of Applied Sciences, Nijmegen, the Netherlands. 2Institute for Management Research, Radboud University, Nijmegen, the Netherlands. 3Open University of the Netherlands, Heerlen, the Netherlands. 4Ghent University, Ghent, Belgium. 5Hubei University, Wuhan, China. 6Kingston University, London, UK. 7Nyenrode Business Universiteit Breukelen Amsterdam the Netherlands Sciences, Nijmegen, the Netherlands. 2Institute for Management Research, Radboud University, Nijmegen, the Netherlands. 3Open University of the Netherlands, Heerlen, the Netherlands. 4Ghent University, Ghent, Belgium. 5Hubei University, Wuhan, China. 6Kingston University, London, UK. 7N d B U B k l A d h N h l d 16. Frank JL, Reibel D, Broderick P, Cantrell T, Metz S. The effectiveness of mindfulness-based stress reduction on educator stress and well-being: results from a pilot study. Mindfulness. 2015;6(2):208–16. https://doi.org/10. 1007/s12671-013-0246-2. bei University, Wuhan, China. 6Kingston University, London, UK. nrode Business Universiteit, Breukelen, Amsterdam, the Netherlan 7Nyenrode Business Universiteit, Breukelen, Amsterdam, the Netherlands. 17. Roeser RW, Schonert-Reichl KA, Jha A, Cullen M, Wallace L, Wilensky R, Oberle E, Thomson K, Taylor C, Harrison J. Mindfulness training and reductions in teachers stress and burnout: results of two randomized, waitlist-control field trials. J Edu Psychol. 2013;105(3):787–804. https://doi. org/10.1037/a0032093. Received: 27 November 2019 Accepted: 19 February 2020 Received: 27 November 2019 Accepted: 19 February 2020 Competing interests The authors declare that they have no competing interests. 15. Jennings PA, Frank JL, Snowberg KE, Coccia MA, Greenberg MT. Improving classroom learning environments by cultivating awareness and resilience in education (CARE): results of a randomized controlled trial. School Psychol Quart. 2013;28(4):374–90. https://doi.org/10.1037/spq0000035. Ethics approval and consent to participate The proposed CRCT has some disadvantages com- pared to an RCT [87]. A CRCT has greater complexity in design and analysis and requires more participants/ teachers, to achieve adequate statistical power. The Ethics Committee Practice-based Research of het HAN University of Ap- plied Sciences and the Medical Ethics Committee (METC) of Radboud Univer- sity Medical Centre, both located in Nijmegen, the Netherlands, approved the research proposal (Registration no. ACPO 07.12/15; File number CMO: 2019–5266). Both committees stated that the research complied with the Page 16 of 18 Janssen et al. Trials (2020) 21:376 Janssen et al. Trials (2020) 21:376 School Psychol. 2012;50:129–45. https://doi.org/10.1016/j.jsp.2011.07.003. Epub 2011. School Psychol. 2012;50:129–45. https://doi.org/10.1016/j.jsp.2011.07.003. 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https://openalex.org/W3203309613	https://www.researchsquare.com/article/rs-131006/v1.pdf?c=1608338863000	English	null	Enhancing 3D Printing Producibility in Polylactic Acid Using Fused Filament Fabrication Fused Deposition Modelling and Machine Learning	Research Square (Research Square)	2,020	cc-by	8,963	Mahmoud Moradi Malayer University M. Saleh Meiabadi École de Technologie Supérieure Mojtaba Karami Moghadam Malayer University Sina Ardabili University of Mohaghegh Ardabili Shahab S. Band National Yunlin University of Science and Technology Amir Mosavi (  amir.mosavi@mailbox.tu-dresden.de ) TU Dresden Mahmoud Moradi Malayer University M. Saleh Meiabadi École de Technologie Supérieure Mojtaba Karami Moghadam Malayer University Sina Ardabili University of Mohaghegh Ardabili Shahab S. Band National Yunlin University of Science and Technology Amir Mosavi (  amir.mosavi@mailbox.tu-dresden.de ) TU Dresden Enhancing 3D Printing Producibility in Polylactic Acid Using Fused Filament Fabrication and Machine Learning Mahmoud Moradi 1, M. Saleh Meiabadi 2, Mojtaba Karami Moghadam 1, Sina Ardabili 3, Shahab S. Band 4, and Amir Mosavi 5, Mahmoud Moradi 1, M. Saleh Meiabadi 2, Mojtaba Karami Moghadam 1, Sina Ardabili 3, Shahab S. Band 4, and Amir Mosavi 5, 1 Department of Mechanical Engineering, Faculty of Engineering, Malayer University, Malayer, Iran 1 Department of Mechanical Engineering, Faculty of Engineering, Malayer University, Malayer, Iran 2 Department of Mechanical Engineering, École de Technologie supérieure, Canada 1100 Notre-Dame West, Montreal, QC, H3C 1K3, Canada; mohammadsaleh.sheikhmohammadmeiabadi.1@ens.etsmtl.ca 3 Biosystem Engineering Department, University of Mohaghegh Ardabili, Ardabil 5619911367, Iran; s.ardabili@ieee.org Department of Mechanical Engineering, Faculty of Engineering, Malayer University, Malayer, Iran Department of Mechanical Engineering, École de Technologie supérieure, Canada 1100 Notre-Dame West, Montreal, QC, H3C 1K3, Canada; mohammadsaleh.sheikhmohammadmeiabadi.1@ens.etsmtl.ca Biosystem Engineering Department University of Mohaghegh Ardabili Ardabil 5619911367 Iran; 2 Department of Mechanical Engineering, École de Technologie supérieure, Canada 1100 Notre-Dame W Montreal, QC, H3C 1K3, Canada; mohammadsaleh.sheikhmohammadmeiabadi.1@ens.etsmtl.ca 2 Department of Mechanical Engineering, École de Technologie supérieure, Canada 1100 Notre-Dame West, Montreal, QC, H3C 1K3, Canada; mohammadsaleh.sheikhmohammadmeiabadi.1@ens.etsmtl.ca 3 Biosystem Engineering Department, University of Mohaghegh Ardabili, Ardabil 5619911367, Iran; s.ardabili@ieee.org 3 Biosystem Engineering Department, University of Mohaghegh Ardabili, Ardabil 5619911367, Iran; s.ardabili@ieee.org 4 Faculty Future Technology Research Center, College of Future, National Yunlin University of Science and Technology, 123 University Road, Section 3, Douliou, Yunlin 64002, Taiwan; shamshirbands@yuntech.edu.tw 4 Faculty Future Technology Research Center, College of Future, National Yunlin University of Science and Technology, 123 University Road, Section 3, Douliou, Yunlin 64002, Taiwan; shamshirbands@yuntech.edu.tw 5 Norwegian University of Life Sciences, 1430 Ås, Norway 4 Faculty Future Technology Research Center, College of Future, National Yunlin University of Science a Technology, 123 University Road, Section 3, Douliou, Yunlin 64002, Taiwan; shamshirbands@yuntech.edu. 5 Norwegian University of Life Sciences, 1430 Ås, Norway Received: date; Accepted: date; Published: date Abstract: Polylactic acid (PLA) is one of the high applicable material which is used in the 3D printers due to some significant features like its deformation property and affordable cost. For improvement of the end-use quality, it is of significant importance to enhance the quality of Fused Filament Fabrication (FFF)-printed objects in PLA. The purpose of this investigation is to boost toughness and to reduce the production cost of the FFF-printed tensile test samples with the desired part thickness. Due to prevent from many numerous and idle printing samples the response Surface Method (RSM) is used. Research Article Keywords: Fused Filament Fabrication, toughness, part thickness, machine learning, arti¦cial neural network, response surface method, arti¦cial intelligence, deep learning, 3D printing, big data, data science, design of experiment, additive manufacturing Posted Date: December 18th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-131006/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License 1. Introduction The industrial companies are gradually adapting to additive manufacturing (AM) technologies due to higher flexibility, less waste material, fewer tooling requirements [1]. Some traces are emerging in a number of industries for the replacement of Traditional Manufacturing (TM) with Additive Manufacturing (AM). FFF is one of the most applicable AM technologies used to fabricate plastic products. FFF printer directly builds 3D parts from a 3D Computer-Aided Design (CAD) model by fusing successive extruded layers of feedstock material that fuse together to produce components layer by layer. y y y Several studies have evaluated FFF process parameters on mechanical properties and dimensional tolerances of printed parts by the design of experiments method [2-6] and by evolutionary algorithm [7]. Qattawi et al. [1] checked the effects of processing criteria on mechanical properties and dimensional accuracy using 18 printed samples. Ceretti et al. [8] statistically analyzed the implementation of the two types of the process criteria and the extrusion head on the dimensions of multi-layered PCL scaffolds and pores in the deposited material using a modified FFF printer. The extrusion heads were wire extrusion and powder extrusion head. Extrusion head type did not strongly influence resulting geometry of the samples. Griffiths et al. [9] used design of experiments method (DOE) to quantify the effects of build. Results indicated that infill percentage and number of shells are significant factors to optimize tensile properties. Moreover, maximum layer thickness and lowest infill percentage, and number of shells have to be used to optimize efficiency outputs. Lieneke et al. [10] developed a method to identify realistic tolerance values for additive manufacturing and factors influencing the geometrical accuracy. The materials, machines, and process parameters for FFF, laser sintering (LS), and Laser Melting (LM) were defined for the development of the method. Rezaie et al. [11] objective were to study implementing a mathematical tool used in the conceptual design stage for topology optimization. They investigated the application of topology optimization for the production of meso-scale structures to realizing intermediated density regions. Mahmood et al. [6] applied Taguchi’s experiment method to test the effects of process parameters on structural definitude and geometric characteristics [7]. The mechanical properties of additively manufactured parts suffer compared to conventionally manufactured parts [12]. PLA presents a relatively brittle behavior under tensile loading [13]. Enhancing 3D Printing Producibility in Polylactic Acid Using Fused Filament Fabrication and Machine Learning The statistical analysis is performed to deal with this concern considering extruder temperature (ET), infill percentage (IP), and layer thickness (LT) as controlled factors. The tensile test specimens are printed based on the designed experiments, and the tensile strength tests are conducted by SANTAM 150 universal testing machine based on ASTM D638. The pattern for filling is designed based on honeycomb which is applied to produce lightweight and high-strength specimens. The area under Force- Extension curve up to fracture is acquired as the toughness of the printed specimens. This study also developed a modeling process using artificial neural network (ANN) and artificial neural network- genetic algorithm (ANN-GA) techniques to develop an accurate estimation for toughness, part thickness, and production cost dependent variables. Results were evaluated by correlation coefficient and RMSE values. According to the modeling results, ANN-GA as a hybrid machine learning (ML) technique could enhance the accuracy of modeling about 7.5, 11.5 and 4.5 % for toughness, part thickness, and production cost, respectively, in comparison with those for the single ANN method. On the other side, the optimization results confirm that the optimized specimen is cost-effective and able to comparatively undergo deformation, which enables the usability of printed PLA objects. The research is accomplished under the constraints of PLA compatibility with existing Fused Filament Fabrication installation, in the absence of the functional assistant of the machine. Although the mechanical properties and dimensional accuracy of PLA have already been studied, there is little literature on the toughness of the printed PLA with honeycomb internal fill pattern. Keywords: Fused Filament Fabrication; toughness; part thickness; machine learning; artificial neural network; response surface method; artificial intelligence; deep learning; 3D printing; big data; data science; design of experiment; additive manufacturing 1. Introduction Although the dimensional accuracy and mechanical properties of PLA have been already studied, there is little literature on the printed PLA's toughness with honeycomb internal fill pattern [14-18]. Hence, effects of extruder temperature (ET), infill percentage (IP), and layer thickness (LT) and their interactions on toughness, thickness, and production cost of the 3D printed specimens in PLA are investigated by Response Surface Methodology (RSM). One reason may more significant about using of RSM in many scientific studies is that this method shows better the interaction between parameters and by graphic diagrams [7]. Recently, Machine learning (ML) techniques have become as one of the effective tools for modeling and simulating scientific phenomena, mechanical properties, engineering processes and different material behaviors in mechanical engineering fields. This section presents the notable studies which employed ML techniques to handle modeling and predicting tasks in FFF 3D printers. Buys et al [14] conducted research on 3D printer for the multi-material structure of the polymeric matrix. In this research, they evaluated mechanical properties of samples such as wear, Flexural, and morphological properties. PLA-PA6/TiO2 polymeric matrix are printed and the wear examination shows that the wear rate for PA6/TiO2 samples is 823 µm and for PLA samples is 1092 µm. Yadav et al [14] employed adaptive neuro-fuzzy inference system (ANFIS) as a hybrid ML technique for the prediction of tensile strength in PETG and ABS in the presence of temperature, material density and layer height as input variables. Results were evaluated by error percentage. According to results the ANFIS could successfully cope with the task by error percentage 2.63. The maximum tensile strength was estimated to be 0.0405 kN/mm2 for PETG in the presence of 0.1 mm layer height, material density 1.27 g/cm3 and extrusion temperature 225 °C. Ali and Chowdary [15] employed ANN for the prediction the mechanical characteristics of FFF printed parts in the presence of air gap, raster angle, number of contours and build orientation as input variables. ANN was trained using a Bayesian function. Results were evaluated by accuracy. According to the results, ANN could successfully cope with the task by enhancing the accuracy by about 5%. Sheoran and Kumar [16] developed a comparative study for analysing GA, Taguchi method, gray relational, RSM, ANN, fractional factorial and Fuzzy logic for handling the FFF approach to enhance the structural specifics as well as printed sample quality. 1. Introduction According to the results, hybrid ML techniques improved the accuracy and increased performance compared to the single ML techniques. According to the literature, the ML techniques can be effective tools for modeling the FFF process [14-17]. In addition, hybrid ML techniques provided higher accuracy and performance compared with single ML techniques [15]. This made us to move toward comparing ANN as the frequently used and simple ML technique with ANN-GA as the hybrid ML technique. Therefore, the objectives of the present work can be categorized into two main stages. The first step is to improve the mechanical behaviour of the FFF printed PLA under tensile loading and reduce the production cost of the specimens. The second step is to estimate the Toughness (N-mm), Part thickness (mm) and Production Cost ($) in the presence of LT, LP and ET using ANN and ANN-GA techniques. The honeycomb internal fill pattern is applied to increase the printed samples' ductility and decrease material use. The area under Force- Extension curve up to fracture is considered the toughness of the printed specimens. The part thickness is measured by a micrometer of 0.01 mm resolution made by Mitutoyo (Mitutoyo company, Model 500-196-30 AOS Absolute Digimatic Caliper, Japan). The production cost is calculated using a formula based on reasonable prices in the FFF 3D printing market. The acquired data is analysed by Design-Expert V8 software via the response surface method. The independent factors are optimized and examined to affirm that the research method is viable. 2.1. Response surface methodology and ANN-GA RSM is based on applied mathematics and the statistical techniques to determine functional relationships between output responses which are affected by input factors [17]. RSM generates an empirical polynomial model of approximation for response surface over a factor region [18]. The smaller the region of interest, the better the approximation. When all the independent factors are continuous and can be estimated and regulated for experimental studies. Thus, the response surface can be presented through the Equation 1 [19]. Y= f(x1, x2, x3, ..., xk) (1) (1) (1) Here, k is the number of independent factors. The approximation of its mathematical model is represented through the infinite strings of x Taylor. A quadratic polynomial function expressed in Equation 2 is implemented in RSM [12,13,20]. 𝑦= 𝛽0 + ∑𝛽𝑖𝑥𝑖 𝑘 𝑖=1 + ∑𝛽𝑖𝑖𝑥𝑖 2 𝑘 𝑖=1 + ∑∑𝛽𝑖𝑗𝑥𝑖 𝑗 𝑖 𝑥𝑗+ 𝜀 (2) (2) In equation 2, β, βi, βii, and βij are constant, linear coefficients, coefficients of quadratic, interaction coefficients, respectively. Furthermore, ε represents the regression error. In equation 2, β, βi, βii, and βij are constant, linear coefficients, coefficients of quadratic, interaction coefficients, respectively. Furthermore, ε represents the regression error. Here, the input factors include extruder temperature, infill percentage layer, and thickness. As discussed by Moradi et al. [19] the data is obtained from an experimental analysis using Design- Expert V8 software. Table 1 shows three factors, i.e., the statistical analysis based on Central Composite Design (CCD), full replication of three agents, and five stages. Based on the previous research [19] for each of factors is set significant domain because higher and lower these ranges the 3D printer has proper efficiency. Toughness (N-mm), part thickness (mm), and production cost ($) have opted as output responses. The samples are printed by FFF printer model Sizan 3 (Sizan company, Kashan, Iran). Table 1. Experimental data for levels of independent factors Factor Unit Levels -2 -1 0 1 2 LT mm 0.1 0.15 0.2 0.25 0.3 IP % 10 20 30 40 50 ET C 190 200 210 220 230 The part thickness is measured by a micrometer of 0.01 mm resolution made by Mitutoyo. The production cost of the specimens is calculated by a formula obtained from 3D printing market. Equation (3) offers the production cost of PLA printed parts in terms of build time and part weight. The design matrix and experiment results are reported in Table 2 [23]. 2.1. Response surface methodology and ANN-GA Production Cost = 0.5 Build time (min) + 0.03 Part weight (gr) (3) (3) Table 2. Experimental data for DOE Run Input Factors Output Responses Type of Fracture LT IP ET Toughness (N-mm) Part thickness (mm) Production Cost ($) 1 0.20 30.00 210.00 1829.27 3.98 17.73 Brittle 2 0.20 30.00 210.00 1394.35 3.84 17.73 Brittle 3 0.15 40.00 220.00 1157.86 3.88 21.72 Brittle 4 0.30 30.00 210.00 5164.36 3.68 13.77 Tough 5 0.20 30.00 210.00 1674.03 4.02 17.73 Brittle 6 0.25 40.00 200.00 5144.17 4.00 15.76 Tough 7 0.25 20.00 200.00 1835.62 3.82 15.25 Brittle 8 0.15 20.00 220.00 2239.94 4.48 20.2 Brittle 9 0.20 30.00 210.00 4112.96 4.04 17.73 Tough 10 0.15 40.00 200.00 1520.79 3.98 21.72 Brittle 11 0.20 30.00 210.00 1140.16 4.08 17.73 Brittle 12 0.20 10.00 210.00 1167.21 3.86 16.72 Brittle 13 0.10 30.00 210.00 830.976 3.98 27.19 Brittle 14 0.15 20.00 200.00 817.052 4.08 20.2 Brittle 15 0.20 30.00 230.00 2644.34 4.08 17.23 Brittle 16 0.20 30.00 190.00 2075.45 3.74 17.23 Brittle 17 0.20 50.00 210.00 2462.57 3.9 18.25 Brittle 18 0.25 40.00 220.00 4489.05 4.12 15.76 Tough 19 0.25 20.00 220.00 5046.5 3.8 15.25 Tough 20 0.20 30.00 210.00 1393.06 3.86 17.73 Brittle Table 2. Experimental data for DOE ANNs are considered as computational intelligence tools inspired by the biological neural networks [7,21]. ANNs train to do tasks by considering the existing mapping of the dataset. The architecture of ANN is based on connections of layers by nodes called neurons and neurons of the biological brain. Each connection transmits a signal from neurons to other neurons, which is like the synapses in a biological brain [22]. The output values of each neuron is affected by weights and bias values. All links between input layers and hidden layers composed the input weight matrix and all links between hidden layers and output layers composed the output weight matrix. Weight (w) which controls the propagation value (x) and the output value (O) from each node is modified using the value from the preceding layer according to Eq. 4 that presents the relation for producing the output values of each neurons [24]. 𝑂= 𝑓(𝑇+ ∑𝑤𝑖𝑥𝑖 𝑛 𝑖=1 ) (4) 𝑂= 𝑓(𝑇+ ∑𝑤𝑖𝑥𝑖 𝑖=1 ) (4) (4) Where T is the specific threshold (bias) value for each node. f is a non-linear sigmoid function, which increases monotonically. The architecture of the proposed ANN is presented in Figure 1. 2.1. Response surface methodology and ANN-GA Where T is the specific threshold (bias) value for each node. f is a non-linear sigmoid function, which increases monotonically. The architecture of the proposed ANN is presented in Figure 1. Input layer Hidden layer Output layer LT IP ET Toughness Production Cost Part thickness Weights Bias Figure 1. The architecture of ANN Input layer Figure 1. The architecture of ANN The training phase is performed by MATLAB software. For the implementation LT, IP and ET are used as input variables for the prediction of Toughness, Part thickness, and Production cost, respectively. 70% of total data were separated randomly for developing the training process by the network. The remaining data were employed for the testing process and evaluating the accuracy of the network. The training phase was initiated using 10 neurons in the hidden layer and continued to 16 neurons by interval 2 neurons. For each step, output data were generated and evaluated by evaluation criteria in comparison with target values. Recently, hybrid methods provided a higher accuracy compared with single techniques [25,26]. These techniques employ a predictor and an optimizer for developing an accurate prediction model. The general mechanism is to employ an optimizer for improving the architecture of the predictor for reaching the best response. One of the frequently used and popular hybrid methods is ANN-GA. A population of candidate solutions to an optimization problem is evolved toward an optimal implementation in the GA. Each candidate solution has a set of properties to reduce the cost function errors. In ANN-GA technique, cost function is the output of layers as a function of weights and bias values. GA employs population and generation sizes as a set of properties and compounds a cost function. The optimization of the cost function aims at reducing the error values. In this case, the error values reduction contributes to providing accurate outputs for the network compared to using a single ANN. Figure 2 represents the flowchart of the proposed machine learning hybrid model of ANN-GA, adapted and reproduced from [27]. To develop ANN architecture Input layer Hidden layer Output layer LT IP ET Toughness Production Cost Part thickness Weights Bias To import weights and bias values to GA To import weights and bias values to GA Start To generate population and generation sizes as a set of properties To evaluate the fitness function Meet the terminating criterion? 2.1. Response surface methodology and ANN-GA NO Yes To generate outputs End Figure 2. The flowchart of ANN-GA developing process Start To develop ANN architecture To import weights and bias values to GA To import weights and bias values to GA To generate population and generation sizes as a set of properties Meet the terminating criterion? To evaluate the fitness function To generate outputs Figure 2. The flowchart of ANN-GA developing process Table 3 presents the evaluation criteria that compare predicted and output values. These factors are also called performance factors that handle the target and output values (the predicted by models). The correlation coefficient is an index for measuring the linearity of target and output values. The root mean square error calculates the deviation error of the output values compared to the target values [28]. These factors are considered the frequently used evaluation metrics in different modeling tasks [29]. Table 3. Model Evaluation metrics Description Accuracy and Performance Index -N is the number of Data - A is the desired output value and P is the output value. Correlation coefficient= 𝑁∑(𝐴 𝑃) −∑(𝐴 ) ∑( 𝑃) √[𝑁∑𝐴2 −(∑𝐴 ) 2][𝑁∑𝑃2 −(∑𝐴𝑃 ) 2] RMSE= √ 1 𝑁∑(𝐴− 𝑃)2 Table 3. Model Evaluation metrics 2.2. Experimental work The tensile test samples fabricated in PLA are investigated mechanically, dimensionally, and economically. Polylactic acids are generated from renewable sources with numerous benefits and divided to some categories, for example the PDLA (Poly-D-lactic Acid), PLLA (Poly-L-lactic Acid), and PDLLA (Poly-DL-lactic Acid) [30]. PLA has a low printing temperature and can be printed both with and without a heated print bed. Material properties of PLA are shown on Table 4. Despite all of these notable characteristics, PLA is brittle and it is not a true choice for items which might be bent, twisted, or dropped. Table 4. Material properties of data sheet [30]. Table 4. Material properties of data sheet [30]. Property Value Full Name Polylactic acid (PLA) Melting Point 150 to 160 °C (302 to 320 °F) Glass Transition 60-65 °C Injection Mold Temperature 178 to 240 °C (353 to 464 °F) Density 1.210–1.430 g·cm−3 Chemical Formula (C3H4O2)n Crystallinity 37% Tensile Modulus 2.7–16 GPa molecular weight (Mw) 112 kg/mol ± 1,733 Polydispersity (MW/MN) 1.65 ± 0.05 Property Full Name Melting Point Glass Transition Injection Mold Temperature Density Chemical Formula Crystallinity Tensile Modulus molecular weight (Mw) Polydispersity (MW/MN) Simplify3D software is employed to fine-tune build parameters of the specimens. Simplify3D includes comprehensive tools to work with 3D printers. The tensile test sample is modeled as a STL file by Solidwork based on international standard ISO 527-2 and imported in Simplify3D. Table 5 illustrations definitions of FFF build parameters that are permanent for all experiments. Table 5. FFF build parameters [23] No Build Parameters Unit Value 1 Nozzle diameter mm 0.45 2 Extrusion width mm 0.45 3 Top solid layer - 6 4 Bottom solid layers - 6 5 Default printing speed mm/min 3600 6 Retraction speed mm/min 1800 7 Outline overlap - Full Honeycomb 8 Interior fill percentage % 15 Table 5. FFF build parameters [23] The infill pattern may significantly affect the strength of the 3D printed part. The honeycomb internal fill pattern is applied for the production of light-weight and high-strength specimens. The honeycomb internal fill adhered to top and bottom solid surfaces offers an excellent rigidity. Figure 3 shows the sample size based on ISO 527-2 standard for the tensile examination. Figure 4 depicts 20%, 30 %, and 40 % full honeycomb infill. Figure 5 presents the 3D printed parts in PLA. Figure 3. 2.2. Experimental work The sizes of samples based on ISO 527-2 standard for the tensile examination Figure 3. The sizes of samples based on ISO 527-2 standard for the tensile examination The SANTAM 150 universal test (SANTAM company, Tehran, Iran) is used to conduct tensile strength tests according to ASTM D638 at the constant rate of 2 mm/min. As the Table 2 indicates the specimens have two types of fracture under in-plane loading. Most of the specimens demonstrate brittle behaviour with no visible deformation before fracture. Only five specimens represent tough fracture and apparent deformation occurs before separation. These specimens have both strength and ductility higher than brittle specimens. brittle behaviour with no visible deformation before fracture. Only five specimens represent to fracture and apparent deformation occurs before separation. These specimens have both strength ductility higher than brittle specimens. (c) (b) (a) Figure 4. Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Figure 5. FFF 3D printed parts of PLA (c) (b) (a) (c) Figure 4. Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Figure 4. Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Figure 4. Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Figure 5. FFF 3D printed parts of PLA Figure 5. FFF 3D printed parts of PLA 3. Results The effects of input factors on outputs can be signified by the analysis of variance (ANOVA) results. The ANOVA is created by assuming that the elements are fixed, not random and the design is crossed, not nested. The software selects polynomial terms in the mathematical model. However, the terms must be significant to refrain from aliasing of the model. Design-Expert calculates statistics such as p-values, lack of fit, Adj R-Squared, and Pred R-Square values to appraise the models. The difference between predicted adjusted R-squared and R-Squared indicates whether the model can reliably be used to interpolate data. If the difference is less than 0.2, then the model is fitting the data and can be used to interpolate data. 3.1. Toughness The analysis of the variance table discloses that LT is the main controlling factor influencing toughness. Interaction between infill percentage and extruder temperature is also crucial. Table 6 depicts the ANOVA results of toughness. The difference between predicted R-squared and adjusted R-squared is 0.042, which affirms that the model can efficiently interpolate data. Table 6. ANOVA for toughness Source Sum of Squares (SOS) Df Mean Square (MS) F-Value (F-v) P-value (P-v) Model 1.694E-003 4 4.235E-004 13.04 <0.0001 LT 1.228E-003 1 1.228E-003 37.81 <0.0001 IP 1.250E-004 1 1.250E-004 3.85 0.0687 ET 8.980E-005 1 8.980E-005 2.76 0.1171 (IP) × (ET) 2.513E-004 1 2.513E-004 7.74 0.0140 Residual 4.872E-004 15 3.248E-005 Lack of Fit (LOF) 1.747E-004 10 1.747E-005 0.28 0.9591 Pure Error (PR) 3.125E-004 5 6.250E-005 Cor Total (CT) 2.181E-003 19 Pred R-Square 0.6747 Adj R-Squared 0.7171 R-Squared 0.7766 Table 6. ANOVA for toughness The equation (5) is predictive model of toughness in terms of coded factors: The equation (5) is predictive model of toughness in terms of coded factors: (Toughness)-0.41 = +0.045-8.760E -003 LT -2.795E-003 IP -2.369E-003 ET +5.605E-003(IP)(ET) (5) (5) The equation (6) is predictive model of toughness in terms of actual values: The equation (6) is predictive model of toughness in terms of actual values: The equation (6) is predictive model of toughness in terms of actual values: (6) (Toughness)-0.41 = +0.49164 -0.17521 LT -0.012049 IP -1.91832E -003 ET +5.60471E-005 (IP)(ET (6) (Toughness)-0.41 = +0.49164 -0.17521 LT -0.012049 IP -1.91832E -003 ET +5.60471E-005 (IP)(ET (6) The relative significance of the factors can be obtained by comparing the coefficients of the factors. Figure 6-a depicts the perturbation plot of toughness. A, B, and C curves illustrate sensitivity of toughness to LT, IP, and ET, respectively. The plot indicates that the toughness of specimens is much more sensitive to LT than other controlled factors. The remarkable point is that IP and ET have a similar influence on the toughness while changing one factor and keeping the others constant. Figure 6-b demonstrates 3D surface plot of toughness in terms of ET and IP. As it displays the tough behavior in the printed PLA can be achieved by two procedures. The first way is to increase extruder temperature and decrease IP at a time. The other way is to increase IP and to decrease ET concurrently. The plausible arguments for the improvement of the toughness by the first way are the enhancement of interlayer adhesion between plastic strings at higher temperature and reduction of the trapped air pockets between the strings at lower IP. Moreover, the time required to build inside sections is considerably dependent on the IP. By increasing IP, the nozzle extrudes more hexagonal pattern lines at the inside sections which takes more time considering the same printing speed for all cases of IP. Therefore, there is less time for heat transfer and variation in LTs using lower IP which results in a better fusion between plastic strings. Figure 6-c depicts 3D surface plot of toughness in terms of LT and ET. The surface plot indicates that increasing LT and LT at a time results in increasing toughness. In a specimen with higher LT, a fewer number of sections are needed to print the part. Therefore, a specimen with a thicker layer consists of less interlayer bonding which are potential places to raise stress concentration and crack propagation. Figure 7 is beneficial to compare interlayer bonding and trapped air using thin and thick LT. In addition, higher LT results in lower heat transfer rates and variation in layer temperatures [23] and consequently, better fusion and adhesion of the extruded layers on the solid layers is expected. Figure 8 demonstrates a schematic of temperature variation in lower and higher LT at the same printing speed. (Toughness)-0.41 = +0.49164 -0.17521 LT -0.012049 IP -1.91832E -003 ET +5.60471E-005 (IP)(ET (6) It is evident that printing PLA at lower temperatures results in poor layer bonding. The 3D surface plot (3D-SP) of toughness in terms of IP and LT is presented in Figure 9. (a) (b) (c) Figure 6. a) Perturbation plot of toughness b) 3D-SP of toughness in terms of ET and IP c) 3D-SP of toughness in phrases of LT and ET (b) (a) (b) (c) (a) (b) (c) Figure 6. a) Perturbation plot of toughness b) 3D-SP of toughness in terms of ET and IP c) 3D-SP of toughness in phrases of LT and ET (b) (a) (c) Figure 6. a) Perturbation plot of toughness b) 3D-SP of toughness in terms of ET and IP c) 3D-SP of toughness in phrases of LT and ET (a) (b) Figure 7. Interlayer bonding and trapped air using (a) lower LT (b) higher LT (a) (b) (b) (a) Figure 7. Interlayer bonding and trapped air using (a) lower LT (b) higher LT (a) (b) Figure 8. Temperature variation in (a) lower LT (b) higher LT Figure 9. 3D-SP of toughness in terms of IP and LT (a) (b) (a) (b) Figure 8. Temperature variation in (a) lower LT (b) higher LT Figure 9. 3D-SP of toughness in terms of IP and LT Figure 9. 3D-SP of toughness in terms of IP and LT The 3D-SP implies that increasing LT and IP leads to an increase in toughness. The IP patterns and IP influence the interior solidity of the printed parts. For uniform stress distribution during the tensile test, hexagonal cells can withstand a mechanical load to impede stress increases on the neighboring cell. Additionally, IP specifies hexagonal cell size, and smaller cell sizes result in higher densities and higher strength. Therefore, it is rational to conceive that higher internal IP results in higher toughness. 3.2 Part thickness The dimensional accuracy of plastic printed parts is affected by many parameters. The melted strings are deposited based on the sliced G-Code file. In addition to the investigated parameters, the part geometry and printing speed may influence the strings' placement and, thus, dimensional accuracy of the part. After it has been deposited out of the nozzle, the plastic behavior depends on plastic temperature, stable temperature, and ambient temperature. Although PLA does not shrink that much, it is essential to study the effects of process parameters on the dimensional accuracy of the printed parts in PLA. The variance table analysis indicates that although all input factors and their interactions influence the part thickness, the interaction between LT and IP is the significant parameter influencing the part thickness. Table 7 demonstrates the ANOVA results of the part thickness. Table 7. ANOVA for thickness Source SOS Df MS F-v P-v Model 0.89 6 0.15 4.46 0.0115 LT 0.20 1 0.20 5.98 0.0294 IP 0.024 1 0.024 0.73 0.4096 E) 0.15 1 0.15 4.48 0.0542 (LT) × (IP) 0.36 1 0.36 10.92 0.0057 (LT) ×(ET) 0.061 1 0.061 1.85 0.1968 (IP) × (ET) 0.092 1 0.092 2.79 0.1185 Residual 0.43 13 0.033 PR 0.049 5 9720E-003 LOF 0.38 8 0.048 4.91 0.0482 CT 1.32 19 Pred R-Square -0.5694 Adj R-Squared 0.5220 R-Squared 0.6730 The Eq. 7 represents anticipating part’s model thickness in terms of coded factors: Thickness = +3.99 - 0.11 LT -0.039 (IP) +0.096 (ET) +0.21(LT)(IP) -0.087 (LT)(ET) -011 (IP)(ET) (7) (7) The Eq. 8 represents anticipating part’s model thickness in terms of actual values: Thickness = -9.04550 + 21.77500 (LT) + 0.13688 (IP) +0.076875 (ET) + 0.42500 (LT)(IP) - 0.17500(LT)(ET) -1.07500E -003 (IP)(ET) (8) Thickness = -9.04550 + 21.77500 (LT) + 0.13688 (IP) +0.076875 (ET) + 0.42500 (LT)(IP) - 0.17500(LT)(ET) -1.07500E -003 (IP)(ET) (8) (8) Figure 10-a shows a perturbation plot of the part thickness. The plot represents that part thickness is very sensitive to change all controlled factors. It can also be realized that the central point of controlled factors (LT= 0.2 mm, IP= 30 %, and ET= 210 °C) is a suitable setting to reach the desired part thickness. Figure 10-b depicts 3D surface plot (3D-SP) of part thickness in terms of LT and IP. 3D-SP of part thickness in phrases of ET and LT is presented in Figure 10-c. (a) (b) (b) (b) (a) (c) Figure 10. a) Perturbation plot of thickness b) 3D-SP of thickness in phrases of LT and IP c) 3D-SP of thickness in phrases of ET and LT (c) (c) Figure 10. a) Perturbation plot of thickness b) 3D-SP of thickness in phrases of LT and IP c) 3D-SP of thickness in phrases of ET and LT 3.3. Production cost 3.3. Production cost The ANOVA illustrates the LT is the most important factor influences the production cost. Table 8 depicts the ANOVA outputs of production cost. Also, the "Adj R-Squared" and "Pred R-Squared" are in excellent agreement. Table 8. ANOVA for production cost Table 8. ANOVA for production cost Source SOS Df MS F-v P-v Model 6.769E-005 5 1.354E-005 1464.91 <0.0001 LT 6.592E-005 1 6.592E-005 7133.12 <0.0001 IP 1.555E-006 1 1.555E-006 168.23 <0.0001 ET 0.000 1 0.000 0.000 1.0000 IP2 4.940E-008 1 4.940E-008 5.35 0.0365 ET2 1.927E-007 1 1.927E-007 20.85 0.0004 PE 0.000 5 0.000 LOF 1.294E-007 9 1.438E-008 Residual 1.294E-007 14 9.241E-009 CT 6.782E-005 19 Pred R-Square 0.9940 Adj R-Squared 0.9974 R-Squared 0.9981 Table 8. ANOVA for production cost The Eq.9 expresses anticipating model of production cost in terms of coded factors: (Production Cost)-1.68 = +7965E-003 + 2.030E-003 (LT) -3.117E-004 (IP) + 4.329E-005 (IP)2 + 8.552E-005 (ET)2 (9) (9) The Eq.10 expresses anticipating model of production cost in terms of actual values: (Production Cost) -1.68 = + 0.038884 + 0.040595 (LT) -5.71489E - 005 (IP) -3.59174E -004 (ET) +4.32947E – 007 (IP)2 +8.55175E-007 (ET)2 (10) (Production Cost) -1.68 = + 0.038884 + 0.040595 (LT) -5.71489E - 005 (IP) -3.59174E -004 (ET) +4.32947E – 007 (IP)2 +8.55175E-007 (ET)2 (10) (10) As the coded equation shows, LT has the highest coefficients among equation terms. Figure 11- a depicts a perturbation plot of production cost. The plot confirms production cost is much more sensitive to LT than other input parameters. Figure 11-b shows the effects of LT and IP on production cost in the form of a 3D surface. Build time has a major impact on the production cost based on the suggested equation. The build time is the sum of extruding time of top and bottom solid surfaces and inside sections. The parameter that defines the number of sections to produce a part is LT, and the parameter that determines the extruding time of inside sections is IP. (a) (a) (b) Figure 11. a) Perturbation plot of production cost b) 3D-SP of production cost in phrases of LT and IP (b) (b) (a) Figure 11. a) Perturbation plot of production cost b) 3D-SP of production cost in phrases of LT and IP 3.4. ANN and ANN-GA Techniques ANN and ANN-GA techniques were performed to develop an accurate model for the prediction of toughness, part thickness, and production cost. In the first step, ANN was developed by 10, 12, 14 and 16 neurons in its single hidden layer for choosing the best number of neurons in the hidden layer in the presence of 70 % of total data. Results were evaluated by correlation coefficient and RMSE values and were tabulated in Table 9. As is clear, the best response is related to neuron number 12. Therefore, the architecture of 3-12-3 was selected as the base ANN’s architecture to be optimized by GA. GA implemented ANN's selected architecture in four treatments (based on our experiences in previous studies). These treatments included population size 50, 100, 150, and 200. The results were tabulated in Table 9. Based on Table 9, Pop. Size 150 with Max generation 360 provides higher accuracy for toughness and production cost and Pop. Size 100 for part thickness compared with other Pop. Sizes. this Pop. Size increased the accuracy by about 9.7, 5.8, and 1.2% respectively for Toughness, Part thickness, and Production Cost compared with single ANN. Table 9. Results for the training phase Output factor ANN Correlation coefficient RMSE ANN-GA Correlation coefficient RMSE No. of neurons Pop size Max gen. Toughness (N-mm) 10 0.7877 924.5529274 50 320 0.9439 633.6373621 12 0.8782 908.0737946 100 210 0.8692 734.6853877 14 0.7964 893.2048644 150 360 0.9642 453.8843405 16 0.8789 694.1594251 200 110 0.9186 654.6824998 Part thickness (mm) 10 0.7671 0.12949 50 320 0.9362 0.045035408 12 0.8788 0.075333178 100 210 0.93 0.042059003 14 0.5173 0.084915266 150 360 0.7768 0.059754932 16 0.6324 0.099691882 200 110 0.8538 0.077821436 Production Cost ($) 10 0.8531 1.960663 50 320 09485 1.288136157 12 0.9636 0.970732923 100 210 0.8956 1.556494043 14 0.8235 3.830106928 150 360 0.9754 1.011613758 16 0.842 2.267871729 200 110 0.9105 1.29979527 Table 9. Results for the training phase By considering the training stage, the elected architectures were employed for the testing stage. The results were tabulated in Table 10. As is clear, the accuracy of testing and training stage for single ANN don’t match, in another word, there is a higher difference between the accuracy of testing and training stages for single ANN method. This makes ANN an untrusted approach. On the other hand, hybrid ANN-GA benefits higher sustainability by comparing testing and training results, which provides almost similar accuracy. Table 10. 3.4. ANN and ANN-GA Techniques Results for the testing phase Output factor ANN Correlation coefficient RMSE ANN-GA Correlation coefficient RMSE No. of neuron s Pop size Max gen Toughness (N-mm) 12 0.91 651.7539629 150 360 0.9791 277.4633823 Part thickness (mm) 0.8911 0.118439425 0.9904 0.036062371 Production Cost ($) 0.938 0.861473905 0.9762 0.569953845 Table 10. Results for the testing phase Table 10. Results for the testing phase Figure 12 presents the plot diagrams for ANN and ANN-GA in the testing stage. This plot presents predicted values on the vertical axis and target values on the horizontal axis. Line T=P is the reference one by one line to determine correlation values. Deviation from this line indicated the error value among target and predicted values. Based on Figure 12, ANN-GA provides a higher correlation for target and expected values compared with those of the single ANN method. The part thickness and production costs are owned the highest accuracy of ANN-GA compared with that for the toughness. (b) ANN-GA (a) ANN Figure 12. Plot diagrams for the testing phase. a) hybrid ANN-GA, b) single ANN Figure 12. Plot diagrams for the testing phase. a) hybrid ANN-GA, b) single ANN Figure 13 presents the deviation from target values for comparing ANN and ANN-GA. These figures contain relative deviation error values for testing data in two categories, including single ANN and hybrid ANN-GA. The horizontal 0 line refers to target values, and columns refer to relative deviations for each predicted testing data from target values. As is clear for all three variables, a single ANN provides a higher deviation from target values compared with the hybrid ANN-GA method. These observations claim that hybrid methods offer higher accuracy and lower error compared with single methods. Figure 13. Deviation from target values for hybrid ANN-GA and single ANN methods Figure 13. Deviation from target values for hybrid ANN-GA and single ANN methods 3.5. Numerical optimization The process parameters are optimized based on a criterion defined in Table 11. The standard aims to increase the toughness, achieve the desired thickness and decrease 3D printed parts' production cost at a time. It is anticipated that the optimized specimen demonstrates tough behavior at the least possible production cost with the desired part thickness. The predicted and the experimental results for the implementation of the optimized process parameters are shown in Table 12. The optimum solution has a high level of desirability. Figure 14 depicts the Force- Extension diagram of the tensile test specimens fabricated by the optimal setting. The optimized specimen's improved toughness is more due to an increase in ductility rather than the specimen's strength. By overlaying contour maps from multiple responses, RSM can be used to find the ideal window of operability. The overlaying contour maps to create ideal printed samples is shown in Figure 15. On each contour map, regions that do not meet the significations are grayed-out [31]. Table 11. Criteria, effective inputs, and responses of each parameter Table 11. Criteria, effective inputs, and responses of each parameter Responses/Parameters Name Goal Low. limit Up. limit Low. Weight Up. Weight Importance Parameters LT is in range 0.1 0.3 1 1 - IP is in range 10 50 1 1 - ET is in range 190 230 1 1 - Responses Criteria Toughness Max. 817 5500 1 1 1 Thickness is goal =4 3.68 4.98 1 1 1 Cost Min. 13.77 27.19 1 1 1 Table 12. experimental validation and predicted optimum outputs Sol. Opt. inputs Desirability Output responses LT IP ET Toughness (N-mm) Thickness (mm) Production Cost ($) 1 0.28 38 222 0.99 Actual 5097.727 3.72 14.77 Predicted 5399.99 4.000 14.372 Error% -5.93% -7.5% 2.23% Figure 14. Extension-Force diagram of the optimized specimen Table 12. experimental validation and predicted optimum outputs Figure 14. Extension-Force diagram of the optimized specimen (a) (b) Fi u e 15 O e layi o tou a i te of (a) LT a d IP (b) IP a d ET (a) (b) Figure 15. Overlaying contour maps in terms of (a) LT and IP (b) IP and ET (a) (b) (b) Figure 15. Overlaying contour maps in terms of (a) LT and IP (b) IP and ET References 1. Qattawi, A.; Alrawi, B.; Guzman, A. 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Multi-layered scaffolds production via Fused Deposition Modeling (FDM) using an open source 3D printer: process parameters optimization for dimensional accuracy and design reproducibility. 4. Conclusions The present work aimed to enhance the production of PLA printed parts via investigation toughness, thickness, and production cost of the tensile test specimens. Also, Training was performed by ANN and ANN-GA techniques for developing an accurate model for the prediction of toughness, part thickness, and production cost. This method is performed by MATLAB software and calculates a superb prediction of output parameters. The tensile test of samples not only broad a deep insight of a main PLA’s features, but also it can present brilliant results of printed samples which are printed by some criteria such as IP, ET and LT. The DOE of this study declined 125 tests to only 20 test which it has a big impact on saving time and produced cost. From the results obtained the following concrete conclusions are made. Although PLA is brittle in nature, the results confirm that it is feasible to improve the printed parts' toughness to develop PLA's end-use mechanical applications. Furthermore, because build time plays a major role in determining production cost, it is possible to reduce production cost without a significant impact on desired properties. It is also concluded that, interaction among LT and IP is the main parameter that has an impact on the thickness of the printed part. It can be conceived that due to little shrinkage of the PLA, extruder temperature has less influence on the dimensional accuracy of the PLA. In addition, the optimized setting to enhance producibility of PLA printed parts is layer thickness = 0.28mm, infill percentage = 34%, and extruder temperature = 222°C. The improved toughness of the optimized specimen is more due to an increase in ductility rather than the strength of the specimen. 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Figures Figures Figure 1 The architecture of ANN Figure 1 Figure 2 The §owchart of ANN-GA developing process Figure 2 The §owchart of ANN-GA developing process The §owchart of ANN-GA developing process The §owchart of ANN-GA developing process Figure 3 The sizes of samples based on ISO 527-2 standard for the tensile examination Figure 4 Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Figure 3 The sizes of samples based on ISO 527-2 standard for the tensile examination The sizes of samples based on ISO 527-2 standard for the tensile examination Figure 4 Figure 4 Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Honeycomb internal pattern (a) 20 % (b) 30 % (c) 40 % Figure 5 FFF 3D printed parts of PLA FFF 3D printed parts of PLA Figure 6 Figure 6 a) Perturbation plot of toughness b) 3D-SP of toughness in terms of ET and IP c) 3D-SP of toughness in phrases of LT and ET a) Perturbation plot of toughness b) 3D-SP of toughness in terms of ET and IP c) 3D-SP of toughness in phrases of LT and ET Figure 7 Interlayer bonding and trapped air using (a) lower LT (b) higher LT Figure 8 Temperature variation in (a) lower LT (b) higher LT Figure 7 Interlayer bonding and trapped air using (a) lower LT (b) higher LT Interlayer bonding and trapped air using (a) lower LT (b) higher LT Figure 8 Figure 9 ure 9 SP of toughness in terms of IP and LT ure 10 Perturbation plot of thickness b) 3D-SP of thickness in phrases of LT and IP c) 3D-SP of thickness in ases of ET and LT 3D-SP of toughness in terms of IP and LT 3D-SP of toughness in terms of IP and LT Figure 10 a) Perturbation plot of thickness b) 3D-SP of thickness in phrases of LT and IP c) 3D-SP of thickne phrases of ET and LT Figure 8 Figure 8 Temperature variation in (a) lower LT (b) higher LT Temperature variation in (a) lower LT (b) higher LT Temperature variation in (a) lower LT (b) higher LT Figure 10 a) Perturbation plot of thickness b) 3D-SP of thickness in phrases of LT and IP c) 3D-SP of thickness in phrases of ET and LT a) Perturbation plot of thickness b) 3D-SP of thickness in phrases of LT and IP c) 3D-SP of thickness in phrases of ET and LT Figure 11 a) Perturbation plot of production cost b) 3D-SP of production cost in phrases of LT and IP Figure 11 a) Perturbation plot of production cost b) 3D-SP of production cost in phrases of LT and IP a) Perturbation plot of production cost b) 3D-SP of production cost in phrases of LT and IP Figure 12 Plot diagrams for the testing phase. a) hybrid ANN-GA, b) single ANN Plot diagrams for the testing phase. a) hybrid ANN-GA, b) single ANN Plot diagrams for the testing phase. a) hybrid ANN-GA, b) single ANN Figure 13 Deviation from target values for hybrid ANN-GA and single ANN me Figure 13 Deviation from target values for hybrid ANN-GA and single ANN methods Deviation from target values for hybrid ANN-GA and single ANN methods Deviation from target values for hybrid ANN-GA and single ANN methods Figure 14 Extension-Force diagram of the optimized specimen Figure 14 Extension-Force diagram of the optimized specimen Extension-Force diagram of the optimized specimen Figure 15 Overlaying contour maps in terms of (a) LT and IP (b) IP and ET Figure 15 Overlaying contour maps in terms of (a) LT and IP (b) IP and ET
https://openalex.org/W2587206624	https://escholarship.org/content/qt5wx1j0wh/qt5wx1j0wh.pdf	English	null	Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC	Nature physics	2,017	cc-by	20,825	UC Irvine UC Irvine Previously Published Works Title Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC Permalink https://escholarship.org/uc/item/5wx1j0wh Journal Nature Physics, 13(9) ISSN 1745-2473 Authors Aaboud, M Aad, G Abbott, B et al. Publication Date 2017-09-01 DOI 10.1038/nphys4208 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Title Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC Permalink https://escholarship.org/uc/item/5wx1j0wh Journal Nature Physics, 13(9) ISSN 1745-2473 Authors Aaboud, M Aad, G Abbott, B et al. Publication Date 2017-09-01 DOI 10.1038/nphys4208 Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Peer reviewed UC Irvine UC Irvine Previously Published Works Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Copyright Information Peer reviewed eScholarship.org Powered by the California Digital Library University of California Powered by the California Digital Library University of California Copyright Information This work is made available under the terms of a Creative Commons Attribution License, available at https://creativecommons.org/licenses/by/4.0/ Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC ATLAS Collaboration† Light-by-light scattering (γ γ →γ γ ) is a quantum-mechanical process that is forbidden in the classical theory of electrodynamics. This reaction is accessible at the Large Hadron Collider thanks to the large electromagnetic ﬁeld strengths generated by ultra-relativistic colliding lead ions. Using 480 µb−1 of lead–lead collision data recorded at a centre-of-mass energy per nucleon pair of 5.02 TeV by the ATLAS detector, here we report evidence for light-by-light scattering. A total of 13 candidate events were observed with an expected background of 2.6 ± 0.7 events. After background subtraction and analysis corrections, the ﬁducial cross-section of the process Pb + Pb (γ γ ) →Pb(∗)+ Pb(∗)γ γ , for photon transverse energy ET >3 GeV, photon absolute pseudorapidity \|η\|<2.4, diphoton invariant mass greater than 6 GeV, diphoton transverse momentum lower than 2 GeV and diphoton acoplanarity below 0.01, is measured to be 70 ± 24 (stat.) ±17 (syst.) nb, which is in agreement with the standard model predictions. O relativistic charged particles can be described by the equivalent photon approximation (EPA)17–19, which is schematically shown in Fig. 1b. The EM fields produced by the colliding Pb nuclei can be treated as a beam of quasi-real photons with a small virtuality of Q2 < 1/R2, where R is the radius of the charge distribution and so Q2 < 10−3 GeV2. Then, the cross-section for the reaction Pb + Pb (γ γ ) →Pb + Pb γ γ can be calculated by convolving the respective photon flux with the elementary cross-section for the process γ γ →γ γ . Since the photon flux associated with each nucleus scales as Z 2, the cross-section is extremely enhanced as compared with proton–proton (pp) collisions. O ne of the key features of Maxwell’s equations is their linearity in both the sources and the fields, from which follows the superposition principle. This forbids eﬀects such as light-by-light (LbyL) scattering, γ γ →γ γ , which is a purely quantum-mechanical process. It was realized in the early history of quantum electrodynamics (QED) that LbyL scattering is related to the polarization of the vacuum1. In the standard model of particle physics, the virtual particles that mediate the LbyL coupling are electrically charged fermions or W ± bosons. In QED, the γ γ →γ γ reaction proceeds at lowest order in the fine-structure constant (αem) via virtual one-loop box diagrams involving fermions (Fig. Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC 1a), which is an O(α4 em ≈3×109) process, making it challenging to test experimentally. Indeed, the elastic LbyL scattering has remained unobserved: even the ultra-intense laser experiments are not yet powerful enough to probe this phenomenon2. relativistic charged particles can be described by the equivalent photon approximation (EPA)17–19, which is schematically shown in Fig. 1b. The EM fields produced by the colliding Pb nuclei can be treated as a beam of quasi-real photons with a small virtuality of Q2 < 1/R2, where R is the radius of the charge distribution and so Q2 < 10−3 GeV2. Then, the cross-section for the reaction Pb + Pb (γ γ ) →Pb + Pb γ γ can be calculated by convolving the respective photon flux with the elementary cross-section for the process γ γ →γ γ . Since the photon flux associated with each nucleus scales as Z 2, the cross-section is extremely enhanced as compared with proton–proton (pp) collisions. In this article, a measurement of LbyL scattering in Pb + Pb collisions at the Large Hadron Collider (LHC) is reported, following the approach recently proposed in ref. 20. The final-state signature of interest is the exclusive production of two photons, Pb + Pb (γ γ ) →Pb(∗)+ Pb(∗)γ γ , where a possible EM excitation of the outgoing ions21 is denoted by (∗). Hence, the expected signature is two photons and no further activity in the central detector, since the Pb(∗) ions escape into the LHC beam pipe. Moreover, it is predicted that the background is relatively low in heavy-ion collisions and is dominated by exclusive dielectron (γ γ →e+e−) production20,22. The misidentification of electrons as photons can occur when the electron track is not reconstructed or the electron emits a hard- bremsstrahlung photon. The fiducial cross-section of the process γ γ →γ γ in Pb + Pb collisions is measured, using a data set recorded at a nucleon–nucleon centre-of-mass energy (√sNN) of 5.02 TeV. This data set was recorded with the ATLAS detector at the LHC in 2015 and corresponds to an integrated luminosity of 480 ± 30 µb−1. In addition to the measured fiducial cross-section, the significance of the observed number of signal candidate events is given, assuming the background-only hypothesis. Powered by the California Digital Library University of California eScholarship.org ARTICLES PUBLISHED ONLINE: 14 AUGUST 2017 \| DOI: 10.1038/NPHYS4208 PUBLISHED ONLINE: 14 AUGUST 2017 \| DOI: 10.1038/NPHYS4208 †A full list of authors and afliations appears at the end of the paper. Evidence for light-by-light scattering in heavy-ion collisions with the ATLAS detector at the LHC LbyL scattering via an electron loop has been precisely, albeit indirectly, tested in measurements of the anomalous magnetic moment of the electron and muon3,4 where it is predicted to contribute substantially, as one of the QED corrections5. The γ γ →γ γ reaction has been measured in photon scattering in the Coulomb field of a nucleus (Delbrück scattering) at fixed photon energies below 7 GeV (refs 6–9). The analogous process, where a photon splits into two photons by interaction with external fields (photon splitting), has been observed in the energy region of 0.1–0.5 GeV (ref. 10). A related process involving only real photons, in which several photons fuse to form an electron–positron pair (e+e−), has been measured in ref. 11. Similarly, the multiphoton Compton scattering, in which up to four laser photons interact with an electron, has been observed12. An alternative way by which LbyL interactions can be studied is by using relativistic heavy-ion collisions. In ‘ultra-peripheral collision’ (UPC) events, with impact parameters larger than twice the radius of the nuclei13,14, the strong interaction does not play a role. The electromagnetic (EM) field strengths of relativistic ions scale with the proton number (Z). For example, for a lead (Pb) nucleus with Z = 82 the field can be up to 1025 V m−1 (ref. 15), much larger than the Schwinger limit16 above which QED corrections become important. In the 1930s it was found that highly Experimental set-up l d l ATLAS is a cylindrical particle detector composed of several sub- detectors23. ATLAS uses a right-handed coordinate system with its origin at the nominal interaction point in the centre of the detector and the z axis along the beam pipe. The x axis points from the interaction point to the centre of the LHC ring, and the y NATURE PHYSICS DOI: 10.1038/NPHYS4208 = X X X Pb82+ Pb82+ X v ≈ c v ≈ −c EM ﬁelds EM ﬁelds Pb Pb a b Figure 1 \| Diagrams illustrating the QED LbyL interaction processes and the equivalent photon approximation. a, Diagrams for Delbrück scattering (left), photon splitting (middle) and elastic LbyL scattering (right). Each cross denotes external ﬁeld legs, for example, an atomic Coulomb ﬁeld or a strong background magnetic ﬁeld. b, Illustration of an ultra-peripheral collision of two lead ions. Electromagnetic interaction between the ions can be described as an exchange of photons that can couple to form a given ﬁnal state X. The ﬂux of photons is determined from the Fourier transform of the electromagnetic ﬁeld of the ion, taking into account the nuclear electromagnetic form factors. X X X a v ≈ c v ≈ −c EM ﬁelds EM ﬁelds Pb Pb b b a Pb82+ Pb82+ X EM ﬁelds EM ﬁelds Figure 1 \| Diagrams illustrating the QED LbyL interaction processes and the equivalent photon approximation. a, Diagrams for Delbrück scattering (left), photon splitting (middle) and elastic LbyL scattering (right). Each cross denotes external ﬁeld legs, for example, an atomic Coulomb ﬁeld or a strong background magnetic ﬁeld. b, Illustration of an ultra-peripheral collision of two lead ions. Electromagnetic interaction between the ions can be described as an exchange of photons that can couple to form a given ﬁnal state X. The ﬂux of photons is determined from the Fourier transform of the electromagnetic ﬁeld of the ion, taking into account the nuclear electromagnetic form factors. axis points upwards. Cylindrical coordinates (r,φ) are used in the transverse plane, with φ being the azimuthal angle around the z axis. The pseudorapidity is defined in terms of the polar angle θ as η=−ln tan(θ/2). theoretical uncertainty on the cross-section is mainly due to limited knowledge of the nuclear electromagnetic form factors and the related initial photon fluxes. This is studied in ref. 20 and the relevant uncertainty is conservatively estimated to be 20%. Higher-order corrections (not included in the calculations) are also part of the theoretical uncertainty and are of the order of a few per cent for diphoton invariant masses below 100 GeV (refs 31,32). Angular distance is measured in units of 1R≡√(1η)2 +(1φ)2. The photon or electron transverse energy is ET =E sin(θ), where E is its energy. NATURE PHYSICS DOI: 10.1038/NPHYS4208 The inner tracking detector (ITD) consists of a silicon pixel system, a silicon microstrip detector and a straw-tube tracker immersed in a 2T magnetic field provided by a superconducting solenoid. The ITD track reconstruction eﬃciency is estimated in ref. 24 for minimum-bias pp events that, like UPC Pb + Pb events, have a low average track multiplicity. For charged hadrons in the transverse momentum range 100 < pT < 200 MeV the eﬃciency is about 50% and grows to 80% for pT > 200 MeV. Around the tracker there is a system of EM and hadronic calorimeters, which use liquid argon and lead, copper or tungsten absorbers for the EM and forward (\|η\| > 1.7) hadronic components of the detector, and scintillator-tile active material and steel absorbers for the central (\|η\| < 1.7) hadronic component. The muon spectrometer consists of separate trigger and high-precision tracking chambers measuring the trajectory of muons in a magnetic field generated by superconducting air-core toroids. The ATLAS minimum-bias trigger scintillators (MBTSs) consist of scintillator slabs positioned between the ITD and the endcap calorimeters with each side having an outer ring of four slabs segmented in azimuthal angle, covering 2.07<\|η\|<2.76 and an inner ring of eight slabs, covering 2.76<\|η\|<3.86. The ATLAS zero-degree calorimeters (ZDCs), located along the beam axis at 140 m from the interaction point on both sides, detect neutral particles (including neutrons emitted from the nucleus). The ATLAS trigger system25 consists of a Level-1 trigger implemented using a combination of dedicated electronics and programmable logic, and a software-based high-level trigger. The sources of background considered in this analysis are: γ γ →e+e−, central exclusive production (CEP) of photon pairs, exclusive production of quark–antiquark pairs (γ γ →q¯q) and other backgrounds that could mimic the diphoton event signatures. The γ γ →e+e−background is modelled with STARlight 1.1 (ref. 30), in which the cross-section is computed by combining the Pb + Pb EPA with the leading-order formula for γ γ →e+e−. This process has been recently measured by the ALICE Collaboration, and a good agreement with STARlight is found33. The exclusive diphoton final state can be also produced via the strong interaction through a quark loop in the exchange of two gluons in a colour-singlet state (see Supplementary Fig. 2). This CEP process, gg →γ γ , is modelled using SUPERCHIC 2.03 (ref. NATURE PHYSICS DOI: 10.1038/NPHYS4208 34), in which the pp cross- section has been scaled by A2R4 g as suggested in ref. 20, where A = 208 and Rg ≈0.7 is a gluon shadowing correction35. This process has a large theoretical uncertainty, of O (100%), mostly related to incomplete knowledge of gluon densities36. The γ γ →q¯q contribution is estimated using Herwig++ 2.7.1 (ref. 37) where the EPA formalism in pp collisions is implemented. The γ γ →q¯q sample is then normalized to the corresponding cross-section in Pb + Pb collisions30. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics 852 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE PHYSICS DOI: 10.1038/NPHYS4208 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS DOI: 10.1038/NPHYS4208 NATURE PHYSICS DOI: 10.1038/NPHYS4208 ET (GeV) 0 2 4 6 8 10 12 14 16 18 Photon PID efficiency 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ATLAS Data, 480 μb−1 MC Data, 480 μb−1 MC Ee T − pT trk2 (GeV) 0 2 4 6 8 10 12 14 16 18 Photon reconstruction efficiency 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ATLAS Pb + Pb √sNN = 5.02 TeV Pb + Pb √sNN = 5.02 TeV b a Figure 2 \| Photon identiﬁcation and reconstruction efciencies. a, Photon PID efciency as a function of photon ET extracted from FSR event candidates. b, Photon reconstruction efciency as a function of photon ET (approximated with Ee T −ptrk2 T ) extracted from γ γ →e+e−events with a hard-bremsstrahlung photon. Data (ﬁlled markers) are compared with MC simulations (open markers). The statistical uncertainties arising from the ﬁnite size of the data and simulation samples are indicated by vertical bars. ET (GeV) 0 2 4 6 8 10 12 14 16 18 Photon PID efficiency 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ATLAS Data, 480 μb−1 MC Pb + Pb √sNN = 5.02 TeV a Data, 480 μb−1 MC Ee T − pT trk2 (GeV) 0 2 4 6 8 10 12 14 16 18 Photon reconstruction efficiency 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 ATLAS Pb + Pb √sNN = 5.02 TeV b Figure 2 \| Photon identiﬁcation and reconstruction efciencies. a, Photon PID efciency as a function of photon ET extracted from FSR event candidates. b, Photon reconstruction efciency as a function of photon ET (approximated with Ee T −ptrk2 T ) extracted from γ γ →e+e−events with a hard-bremsstrahlung photon. Data (ﬁlled markers) are compared with MC simulations (open markers). The statistical uncertainties arising from the ﬁnite size of the data and simulation samples are indicated by vertical bars. sum of cluster transverse energies (Ecl1 T +Ecl2 T ). The eﬃciency grows from about 70% at (Ecl1 T +Ecl2 T ) = 6 GeV to 100% at (Ecl1 T +Ecl2 T ) > 9 GeV. The eﬃciency is parameterized using an error function fit, which is then used to reweight the simulation. Performance and validation of photon reconstruction Since the analysis requires the presence of low-energy photons, which are not typically used in ATLAS analyses, detailed studies of photon reconstruction and calibration are performed. The photon particle identification (PID) in this analysis is based on three shower-shape variables: the lateral width of the shower in the middle layer of the EM calorimeter, the ratio of the energy diﬀerence associated with the largest and second largest energy deposits to the sum of these energies in the first layer, and the fraction of energy reconstructed in the first layer relative to the total energy of the cluster. Only photons with ET >3 GeV and \|η\|<2.37, excluding the calorimeter transition region 1.37 < \|η\| < 1.52, are considered. The pseudorapidity requirement ensures that the pho- ton candidates pass through regions of the EM calorimeter where the first layer is segmented into narrow strips, allowing for good separation between genuine prompt photons and photons coming from the decay of neutral hadrons. A constant photon PID eﬃciency of 95% as a function of η with respect to reconstructed photon can- didates is maintained. This is optimized using multivariate analysis techniques40, such that EM energy clusters induced by cosmic-ray muons are rejected with 95% eﬃciency. High-pT γ γ →ℓ+ℓ−production with a final-state radiation (FSR) photon is used for the measurement of the photon PID eﬃciency. Events with a photon and two tracks corresponding to oppositely charged particles with pT >1 GeV are required to pass the same trigger as in the diphoton selection or the supporting trigger. The 1R between a photon candidate and a track is required to be greater than 0.2 to avoid leakage of the electron clusters from the γ γ →e+e−process to the photon cluster. The FSR event candidates are identified using a p ttγ T < 1 GeV requirement, where p ttγ T is the transverse momentum of the three-body system consisting of two charged-particle tracks and a photon. The FSR photons are then used to extract the photon PID eﬃciency, which is defined as the probability for a reconstructed photon to satisfy the identification criteria. Figure 2a shows the photon PID eﬃciencies in data and simulation as a function of reconstructed photon ET. Within their statistical precision the two results agree. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS DOI: 10.1038/NPHYS4208 Due to the extremely low noise, very high hit reconstruction eﬃciency and low conversion probability of signal photons in the pixel detector (around 10%), the uncertainty due to the requirement for minimal activity in the ITD is negligible. The MBTS veto eﬃciency was studied using γ γ →ℓ+ℓ−events (ℓ= e, µ) passing a supporting trigger and it is estimated to be (98 ± 2)%. is imposed. This requirement further reduces the fake-photon background from the dielectron final state by a factor of 25, according to simulation. It has almost no impact on γ γ →γ γ signal events, since the probability of photon conversion in the pixel detector is relatively small and converted photons are suppressed at low ET (3–6 GeV) by the photon selection requirements. According to MC studies, the photon selection requirements remove about 10% of low-ET photons. To reduce other fake-photon backgrounds (for example, cosmic-ray muons), the transverse momentum of the diphoton system (p γ γ T ) is required to be below 2 GeV. To reduce background from CEP gg →γ γ reactions, an additional requirement on diphoton acoplanarity, Aco = 1 −1φγ γ/π < 0.01, is imposed. This requirement is optimized to retain a high signal eﬃciency and reduce the CEP background significantly, since the transverse momentum transferred by the photon exchange is usually much smaller than that due to the colour-singlet-state gluons41. Photons are reconstructed from EM clusters in the calorimeter and tracking information provided by the ITD, which allows the identification of photon conversions. Selection requirements are applied to remove EM clusters with a large amount of energy from poorly functioning calorimeter cells, and a timing requirement is made to reject out-of-time candidates. An energy calibration specifically optimized for photons38 is applied to the candidates to account for upstream energy loss and both lateral and longitudinal shower leakage. A dedicated correction39 is applied for photons in MC samples to correct for potential mismodelling of quantities that describe the properties (‘shapes’) of the associated EM showers. Performance and validation of photon reconstruction Since the analysis requires the presence of low-energy photons, which are not typically used in ATLAS analyses, detailed studies of photon reconstruction and calibration are performed. Event selection Candidate diphoton events were recorded in the Pb + Pb run in 2015 using a dedicated trigger for events with moderate activity in the calorimeter but little additional activity in the entire detector. At Level-1 the total ET registered in the calorimeter after noise suppression was required to be between 5 and 200 GeV. Then at the high-level trigger, events were rejected if more than one hit was found in the inner ring of the MBTS (MBTS veto) or if more than ten hits were found in the pixel detector. Monte Carlo simulation and theoretical predictions Several Monte Carlo (MC) samples are produced to estimate background contributions and corrections to the fiducial measurement. The detector response is modelled using a simulation based on GEANT4 (refs 26,27). The data and MC simulated events are passed through the same reconstruction and analysis procedures. The eﬃciency of the Level-1 trigger is estimated with γ γ →e+e− events passing an independent supporting trigger. This trigger is designed to select events with mutual dissociation of Pb nuclei and small activity in the ITD. It is based on a coincidence of signals in both ZDC sides and a requirement on the total ET in the calorimeter below 50 GeV. Event candidates are required to have only two reconstructed tracks and two EM energy clusters. Furthermore, to reduce possible backgrounds, each pair of clusters (cl1, cl2) is required to have a small acoplanarity (1 −1φcl1,cl2/π < 0.2). The extracted Level-1 trigger eﬃciency is provided as a function of the LbyL signal events are generated taking into account box diagrams with charged leptons and quarks in the loops, as detailed in ref. 28. The contributions from W-boson loops are omitted in the calculations since they are mostly important for diphoton masses mγ γ >2mW (ref. 29). The calculations are then convolved with the Pb + Pb EPA spectrum from the STARlight 1.1 MC generator30. Next, various diphoton kinematic distributions are cross-checked with predictions from ref. 20 and good agreement is found. The 853 ARTICLES ARTICLES Performance and validation of photon reconstruction The photon reconstruction eﬃciency is extracted from data using γ γ →e+e−events where one of the electrons emits a hard-bremsstrahlung photon due to interaction with the material of the detector. Events with exactly one identified electron, two reconstructed charged-particle tracks and exactly one photon are studied. The electron ET is required to be above 5 GeV and the pT j y Preselected events are required to have exactly two photons satisfying the above selection criteria, with a diphoton invariant mass greater than 6 GeV. To reduce the dielectron background, a veto on the presence of any charged-particle tracks (with pT >100 MeV, \|η\| < 2.5 and at least one hit in the pixel detector) NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics NATURE PHYSICS DOI: 10.1038/NPHYS4208 Good agreement between data and γ γ →e+e− MC simulation is observed and the photon reconstruction eﬃciency is measured with a 5–10% relative uncertainty at low ET (3–6 GeV). to the first one, except exactly two tracks are required (Ntrk = 2). Good agreement is observed between data and MC simulation in both control regions, but the precision is limited by the number of events in data. A conservative uncertainty of 25% is therefore assigned to the γ γ →e+e−background estimation, which reflects the statistical uncertainty of data in the Ntrk = 1 control region. The contribution from a related QED process, γ γ →e+e−γ γ , is evaluated using the MadGraph5_aMC@NLO MC generator43 and is found to be negligible. The Aco < 0.01 requirement significantly reduces the CEP gg →γ γ background. However, the MC prediction for this process has a large theoretical uncertainty; hence, an additional data-driven normalization is performed in the region Aco>b, where b is a value greater than 0.01 which can be varied. Three values of b (0.01, 0.02, 0.03) are used, where the central value b=0.02 is chosen to derive the nominal background prediction and the values b = 0.01 and b = 0.03 to define the systematic uncertainty. The normalization is performed using the condition: f norm,b gg→γ γ = (Ndata (Aco > b) −Nsig (Aco >b) −Nγ γ →e+e−(Aco>b))/Ngg→γ γ (Aco >b), for each value of b, where Ndata is the number of observed events, Nsig is the expected number of signal events, Nγ γ →e+e−is the expected background from γ γ →e+e−events and Ngg→γ γ is the MC estimate of the expected background from CEP gg →γ γ events. The normalization factor is found to be f norm gg→γ γ = 0.5 ± 0.3 and the background due to CEP gg →γ γ is estimated to be f norm gg→γ γ ×Ngg→γ γ (Aco < 0.01) = 0.9 ± 0.5 events. To verify the CEP gg →γ γ background estimation method, energy deposits in the ZDC are studied for events before the Aco selection. It is expected that the outgoing ions in CEP events predominantly dissociate, which results in the emission of neutrons detectable in the ZDC20. NATURE PHYSICS DOI: 10.1038/NPHYS4208 γγ acoplanarity 0.00 0.01 0.02 0.03 0.04 0.05 0.06 Events/0.005 0 2 4 6 8 10 12 14 Signal selection no Aco requirement Data, 480 μb−1 γγ → γγ MC γγ → e+e− MC CEP γγ MC mγγ (GeV) 0 5 10 15 20 25 30 Events/3 GeV 0 2 4 6 8 10 12 Signal selection with Aco < 0.01 ATLAS Pb + Pb √sNN = 5.02 TeV Data, 480 μb−1 γγ → γγ MC γγ → e+e− MC CEP γγ MC ATLAS Pb + Pb √sNN = 5.02 TeV b a Figure 3 \| Kinematic distributions for γ γ →γ γ event candidates. a, Diphoton acoplanarity before applying the Aco < 0.01 requirement. b, Diphoton invariant mass after applying the Aco < 0.01 requirement. Data (points) are compared to MC predictions (histograms). The statistical uncertainties on the data are shown as vertical bars. γγ acoplanarity 0.00 0.01 0.02 0.03 0.04 0.05 0.06 Events/0.005 0 2 4 6 8 10 12 14 Signal selection no Aco requirement Data, 480 μb−1 γγ → γγ MC γγ → e+e− MC CEP γγ MC ATLAS Pb + Pb √sNN = 5.02 TeV a mγγ (GeV) 0 5 10 15 20 25 30 Events/3 GeV 0 2 4 6 8 10 12 Signal selection with Aco < 0.01 Data, 480 μb−1 γγ → γγ MC γγ → e+e− MC CEP γγ MC ATLAS Pb + Pb √sNN = 5.02 TeV b a Events/0.005 Figure 3 \| Kinematic distributions for γ γ →γ γ event candidates. a, Diphoton acoplanarity before applying the Aco < 0.01 requirement. b, Diphoton invariant mass after applying the Aco < 0.01 requirement. Data (points) are compared to MC predictions (histograms). The statistical uncertainties on the data are shown as vertical bars. of the track that is unmatched with the electron (trk2) is required to be below 2 GeV. The additional hard-bremsstrahlung photon is expected to have E γ T ≈(Ee T −ptrk2 T ). The ptrk2 T < 2 GeV requirement ensures a suﬃcient 1R separation between the expected photon and the second electron, extrapolated to the first layer of the EM calorimeter. The data sample contains 247 γ γ →e+e−events that are used to extract the photon reconstruction eﬃciency, which is presented in Fig. 2b. NATURE PHYSICS DOI: 10.1038/NPHYS4208 Good agreement between the normalized CEP gg →γ γ MC expectation and the observed events with a ZDC signal corresponding to at least 1 neutron is observed in the full Aco range (see Supplementary Information for details). In addition, a cross-check is performed on Z →µ+µ−γ events identified in pp collision data from 2015 corresponding to an inte- grated luminosity of 1.6 fb−1. The results support (in a similar way to ref. 42) the choice to use the three shower-shape variables in this photon PID selection in an independent sample of low-ET photons. The photon cluster energy resolution is extracted from data using γ γ →e+e−events. The electrons from the γ γ →e+e− reaction (see Supplementary Information) are well balanced in their transverse momenta, with very small standard deviation, σpe+ T −pe− T < 30 MeV, much smaller than the expected EM calorimeter energy resolution. Therefore, by measuring (Ecl1 T −Ecl2 T ) distributions in γ γ →e+e−events, one can extract the cluster energy resolution, σEcl T . For electrons with ET < 10 GeV, the σEcl T /Ecl T is observed to be approximately 8% both in data and simulation. An uncertainty of δσEγ T /σEγ T = 15% is assigned to the simulated photon energy resolution and takes into account diﬀerences between σEcl T in data and σEγ T in simulation. T Similarly, the EM cluster energy scale can be studied using the (Ecl1 T +Ecl2 T ) distribution. It is observed that the simulation provides a good description of this distribution, within the relative uncertainty of 5% that is assigned to the EM cluster energy-scale modelling. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics 854 ARTICLES NATURE PHYSICS DOI: 10.1038/NPHYS4208 NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. \| \| \| / p y © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Background estimation Selection γ γ →e+e− CEP gg→γ γ Hadronic fakes Other fakes Total background Signal Data Preselection 74 4.7 6 19 104 9.1 105 Ntrk =0 4.0 4.5 6 19 33 8.7 39 pγ γ T <2 GeV 3.5 4.4 3 1.3 12.2 8.5 21 Aco < 0.01 1.3 0.9 0.3 0.1 2.6 7.3 13 Uncertainty 0.3 0.5 0.3 0.1 0.7 1.5 The signal simulation is based on calculations from ref. 28. In addition, the uncertainties on the expected number of events passing all selection requirements are given. give a negligible contribution to the signal region. The contribution from bottomonia production (for example, γ γ →ηb →γ γ or γ Pb →Υ →γ ηb →3γ ) is calculated using parameters from refs 46, 47 and is found to be negligible. Table 2 \| Summary of systematic uncertainties. Source of uncertainty Relative uncertainty Trigger 5% Photon reco. efciency 12% Photon PID efciency 16% Photon energy scale 7% Photon energy resolution 11% Total 24% The table shows the relative systematic uncertainty on detector correction factor C broken into its individual contributions. The total is obtained by adding them in quadrature. Table 2 \| Summary of systematic uncertainties. Source of uncertainty Relative uncertainty Trigger 5% Photon reco. efciency 12% Photon PID efciency 16% Photon energy scale 7% Photon energy resolution 11% Total 24% The table shows the relative systematic uncertainty on detector correction factor C broken into its individual contributions. The total is obtained by adding them in quadrature. Table 2 \| Summary of systematic uncertainties. Table 2 \| Summary of systematic uncertainties. The contribution from other fake diphoton events (for example those induced by cosmic-ray muons) is estimated using photons that fail to satisfy the longitudinal shower-shape requirement. The total background due to other fake photons is found to be 0.1 ± 0.1 events. As a further cross-check, additional activity in the muon spectrometer is studied. It is observed that out of 18 events satisfying the inverted p γ γ T requirement, 13 have at least one additional reconstructed muon. In the region p γ γ T <2 GeV, no events with muon activity are found, which is compatible with the above- mentioned estimate of 0.1 ± 0.1. The table shows the relative systematic uncertainty on detector correction factor C broken into its individual contributions. The total is obtained by adding them in quadrature. luminosity. Background estimation Low-pT dijet events can produce multiple π0 mesons, which could potentially mimic diphoton events. The event selection requirements are eﬃcient in rejecting such events, and based on studies performed with a supporting trigger, the background from hadronic processes is estimated to be 0.3 ± 0.3 events. MC studies show that the background from γ γ →q¯q processes is negligible. Low-pT dijet events can produce multiple π0 mesons, which could potentially mimic diphoton events. The event selection requirements are eﬃcient in rejecting such events, and based on studies performed with a supporting trigger, the background from hadronic processes is estimated to be 0.3 ± 0.3 events. MC studies show that the background from γ γ →q¯q processes is negligible. Exclusive neutral two-meson production can be a potential source of background for LbyL events, mainly due to their back-to- back topology being similar to that of the CEP gg →γ γ process. The cross-section for this process is calculated to be below 10% of the CEP gg →γ γ cross-section44,45 and it is therefore considered to Due to its relatively high rate, the exclusive production of electron pairs (γ γ →e+e−) can be a source of fake diphoton events. The contribution from the dielectron background is estimated using γ γ →e+e−MC simulation (which gives 1.3 events) and is verified using the following data-driven technique. Two control regions are defined that are expected to be dominated by γ γ →e+e− backgrounds. The first control region is defined by requiring events with exactly one reconstructed charged-particle track and two identified photons that satisfy the same preselection criteria as for the signal definition. The second control region is defined similarly g g g Exclusive neutral two-meson production can be a potential source of background for LbyL events, mainly due to their back-to- back topology being similar to that of the CEP gg →γ γ process. The cross-section for this process is calculated to be below 10% of the CEP gg →γ γ cross-section44,45 and it is therefore considered to 855 ARTICLES NATURE PHYSICS DOI: 10.1038/NPHYS4208 Table1 \| Thenumberofeventsacceptedbythesequentialselectionrequirementsfordata,comparedwiththenumberofbackground and signal events expected from the simulation. Table1 \| Thenumberofeventsacceptedbythesequentialselectionrequirementsfordata,comparedwiththenumberofbackground and signal events expected from the simulation. Table1 \| Thenumberofeventsacceptedbythesequentialselectionrequirementsfordata,comparedwiththenumberofbackground and signal events expected from the simulation. Background estimation The factor C is used to correct for the net eﬀect of the trigger eﬃciency, the diphoton reconstruction and PID eﬃciencies, as well as the impact of photon energy and angular resolution. It is defined as the ratio of the number of generated signal events satisfying the selection criteria after particle reconstruction and detector simulation to the number of generated events satisfying the fiducial criteria before reconstruction. The value of C and its total uncertainty is determined to be 0.31 ± 0.07. The dominant systematic uncertainties come from the uncertainties on the photon reconstruction and identification eﬃciencies. Other minor sources of uncertainty are the photon energy scale and resolution uncertain- ties and trigger eﬃciency uncertainty. To check for a potential model dependence, calculations from ref. 28 are compared with predictions from ref. 20, and a negligible impact on the C-factor uncertainty is found. Table 2 lists the separate contributions to the systematic uncertainty. The uncertainty on the integrated luminosity is 6%. It is derived following a methodology similar to that detailed in refs 49,50, from a calibration of the luminosity scale using x–y beam-separation scans performed in December 2015. The contribution from UPC events where both nuclei emit a bremsstrahlung photon is estimated using calculations from ref. 13 and is found to be negligible for photons with \|η\| < 2.4 and ET >3 GeV. Conclusion In summary, this article presents evidence for the scattering of LbyL in quasi-real photon interactions from 480 µb−1 of ultra-peripheral Pb + Pb collisions at √sNN =5.02 TeV by the ATLAS experiment at the LHC. The statistical significance against the background-only hypothesis is found to be 4.4 standard deviations. After background subtraction and analysis corrections, the fiducial cross-section for the Pb + Pb (γ γ ) →Pb(∗)+ Pb(∗)γ γ process was measured and is compatible with standard model predictions. The cross-section for the Pb + Pb (γ γ ) →Pb(∗)+ Pb(∗)γ γ process is measured in a fiducial phase space defined by the pho- ton transverse energy ET >3 GeV, photon absolute pseudorapidity \|η\| < 2.4, diphoton invariant mass greater than 6 GeV, diphoton transverse momentum lower than 2 GeV and diphoton acoplanarity below 0.01. Experimentally, the fiducial cross-section is given by The analysis is mostly limited by the amount of data available and the lower limit on transverse energy for reconstructed photons (ET =3 GeV), below which more signal is expected. Advancements on these two points would also allow for reconstruction of low-mass mesons decaying into two photons, which in turn could be used to improve detector calibration. The heavy-ion data yield is expected to double at the end of 2018 (and again increase tenfold after σfid = Ndata −Nbkg C × R Ldt (1) (1) where Ndata is the number of selected events in data, Nbkg is the expected number of background events and R Ldt is the integrated Results Photon kinematic distributions for events satisfying the selection criteria are shown in Fig. 3. The shape of the diphoton acoplanarity distribution for γ γ →e+e−events in Fig. 3a reflects the trajectories of the electron and positron in the detector magnetic field, before they emit hard photons in their collisions with the ITD material. In total, 13 events are observed in data whereas 7.3 signal events and 2.6 background events are expected. In general, good agreement bet- ween data and MC simulation is observed. The eﬀect of sequential selection requirements on the number of events selected is shown in Table 1, for each of the data, signal and background samples. g g To quantify an excess of events over the background expectation, a test statistic based on the profile likelihood ratio48 is used. The p value for the background-only hypothesis, defined as the probability for the background to fluctuate and give an excess of events as large or larger than that observed in the data, is found to be 5 × 10−6. 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The experimental data that support the findings of this study are available in HEPData with the identifier http://dx.doi.org/10.17182/hepdata.77761. 32. Kłusek-Gawenda, M., Schäfer, W. & Szczurek, A. Two-gluon exchange contribution to elastic γ γ →γ γ scattering and production of two-photons in ultraperipheral ultrarelativistic heavy ion and proton-proton collisions. Phys. Lett. B 761, 399–407 (2016). Received 9 February 2017; accepted 15 June 2017; published online 14 August 2017 Received 9 February 2017; accepted 15 June 2017; published online 14 August 2017 NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics 856 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Additional information Canada, FQRNT, and the Ontario Innovation Trust, Canada; EPLANET, ERC, ERDF, FP7, Horizon 2020 and Marie Skłodowska-Curie Actions, European Union; Investissements d’Avenir Labex and Idex, ANR, Région Auvergne and Fondation Partager le Savoir, France; DFG and AvH Foundation, Germany; Herakleitos, Thales and Aristeia programmes co-financed by EU-ESF and the Greek NSRF; BSF, GIF and Minerva, Israel; BRF, Norway; CERCA Programme Generalitat de Catalunya, Generalitat Valenciana, Spain; the Royal Society and Leverhulme Trust, United Kingdom. The crucial computing support from all WLCG partners is acknowledged gratefully, in particular from CERN, the ATLAS Tier-1 facilities at TRIUMF (Canada), NDGF (Denmark, Norway, Sweden), CC-IN2P3 (France), KIT/GridKA (Germany), INFN-CNAF (Italy), NL-T1 (Netherlands), PIC (Spain), ASGC (Taiwan), RAL (UK) and BNL (USA), the Tier-2 facilities worldwide and large non-WLCG resource providers. Major contributors of computing resources are listed in ref. 51. Supplementary information is available in the online version of the paper. Reprints and permissions information is available online at www.nature.com/reprints. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional aﬃliations. Correspondence and requests for materials should be addressed to ATLAS Collaboration. Acknowledgements heavy-ion collisions. Phys. Rev. Lett. 118, 171801 (2017) We thank CERN for the very successful operation of the LHC, as well as the support staﬀ from our institutions without whom ATLAS could not be operated eﬃciently. We acknowledge the support of ANPCyT, Argentina; YerPhI, Armenia; ARC, Australia; BMWFW and FWF, Austria; ANAS, Azerbaijan; SSTC, Belarus; CNPq and FAPESP, Brazil; NSERC, NRC and CFI, Canada; CERN; CONICYT, Chile; CAS, MOST and NSFC, China; COLCIENCIAS, Colombia; MSMT CR, MPO CR and VSC CR, Czech Republic; DNRF and DNSRC, Denmark; IN2P3-CNRS, CEA-DSM/IRFU, France; GNSF, Georgia; BMBF, HGF and MPG, Germany; GSRT, Greece; RGC, Hong Kong SAR, China; ISF, I-CORE and Benoziyo Center, Israel; INFN, Italy; MEXT and JSPS, Japan; CNRST, Morocco; FOM and NWO, Netherlands; RCN, Norway; MNiSW and NCN, Poland; FCT, Portugal; MNE/IFA, Romania; MES of Russia and NRC KI, Russian Federation; JINR; MESTD, Serbia; MSSR, Slovakia; ARRS and MIZŠ, Slovenia; DST/NRF, South Africa; MINECO, Spain; SRC and Wallenberg Foundation, Sweden; SERI, SNSF and Cantons of Bern and Geneva, Switzerland; MOST, Taiwan; TAEK, Turkey; STFC, United Kingdom; DOE and NSF, United States of America. In addition, individual groups and members have received support from BCKDF, the Canada Council, CANARIE, CRC, Compute 23. ATLAS Collaboration. The ATLAS experiment at the CERN large hadron collider. JINST 3, S08003 (2008). 24. ATLAS Collaboration. Charged-particle distributions at low transverse momentum in √s=13 TeV pp interactions measured with the ATLAS detector at the LHC. Eur. Phys. J. C 76, 502 (2016). 25. ATLAS Collaboration. Performance of the ATLAS trigger system in 2010. Eur. Phys. J. C 72, 1849 (2012). 26. Agostinelli, S. et al. GEANT4: a Simulation toolkit. Nucl. Instrum. Meth. A 506 250–303 (2003). 27. ATLAS Collaboration. The ATLAS simulation infrastructure. Eur. Phys. J. C 70, 823–874 (2010). 28. Kłusek-Gawenda, M., Lebiedowicz, P. & Szczurek, A. Light-by-light scattering in ultraperipheral Pb-Pb collisions at energies available at the CERN Large Hadron Collider. Phys. Rev. C 93, 044907 (2016). 857 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS \| VOL 13 \| SEPTEMBER 2017 \| www.nature.com/naturephysics Competing ﬁnancial interests The authors declare no competing financial interests. 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. 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Author contributions All authors have contributed to the publication, being variously involved in the design and the construction of the detectors, in writing software, calibrating subsystems, operating the detectors and acquiring data, and finally analysing the processed data. The ATLAS Collaboration members discussed and approved the scientific results. The manuscript was prepared by a subgroup of authors appointed by the collaboration and subject to an internal collaboration-wide review process. All authors reviewed and approved the final version of the manuscript. 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/. 858 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ARTICLES NATURE PHYSICS \| www.nature.com/naturephysics © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. ATLAS Collaboration M. Aaboud181, G. Aad116, B. Abbott145, J. Abdallah10, O. Abdinov14‡, B. Abeloos149, S. H. Abidi210, O. S. AbouZeid184, N. L. Abraham200, H. Abramowicz204, H. Abreu203, R. Abreu148, Y. Abulaiti196,197, B. S. Acharya218,219,236, S. Adachi206, L. Adamczyk61, J. Adelman140, M. Adersberger131, T. Adye171, A. A. Afolder184, T. Agatonovic-Jovin16, C. Agheorghiesei39, J. A. Aguilar-Saavedra160,165, S. P. Ahlen30, F. Ahmadov95,b, G. Aielli174,175, S. Akatsuka98, H. Akerstedt196,197, T. P. A. Åkesson112, A. V. Akimov127, G. L. Alberghi27,28, J. Albert225, M. J. Alconada Verzini101, M. Aleksa46, I. N. Aleksandrov95, C. Alexa38, G. Alexander204, T. Alexopoulos12, M. 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Wang145, R. Wang8, S. M. Wang202, T. Wang57, W. Wang202,aw, W. Wang53, Z. Wang55, C. Wanotayaroj148, A. Warburton118, C. P. Ward44, D. R. Wardrope109, A. Washbrook70, P. M. Watkins21, A. T. Watson21, M. F. Watson21, G. Watts185, S. Watts115, B. M. Waugh109, A. F. Webb13, S. Webb114, M. S. Weber20, S. W. Weber230, S. A. Weber45, J. S. Webster8, A. R. Weidberg152, B. Weinert91, J. Weingarten80, C. Weiser72, H. Weits139, P. S. Wells46, T. Wenaus36, T. Wengler46, S. Wenig46, N. Wermes29, © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS DOI: 10.1038/NPHYS4208 A. Seiden184, J. M. Seixas32, G. Sekhniaidze135, K. Sekhon120, S. J. Sekula64, N. Semprini-Cesari27,28, C. Serfon151, L. Serin149, L. Serkin218,219, M. Sessa176,177, R. Seuster225, H. Severini145, T. Sﬁligoj106, F. Sforza46, A. Sfyrla73, E. Shabalina80, A. Seiden184, J. M. Seixas32, G. Sekhniaidze135, K. Sekhon120, S. J. Sekula64, N. Semprini-Cesari27,28, C. Serfon151, L. Serin149, L. Serkin218,219, M. Sessa176,177, R. Seuster225, H. Severini145, T. Sﬁligoj106, F. Sforza46, A. Sfyrla73, E. Shabalina80, A. Seiden , J. M. Seixas , G. Sekhniaidze , K. Sekhon , S. J. Sekula , N. Semprini-Cesari , C. Serfon , L. Serin L. Serkin218,219, M. Sessa176,177, R. Seuster225, H. Severini145, T. Sﬁligoj106, F. Sforza46, A. Sfyrla73, E. Shabalina80, N. W. Shaikh196,197, L. Y. Shan50, R. Shang222, J. T. Shank30, M. Shapiro18, P. B. Shatalov128, K. Shaw218,219, S. M. Shaw115, A. Shcherbakova196,197, C. Y. Shehu200, Y. Shen145, P. Sherwood109, L. Shi202,an, S. Shimizu97, C. O. Shimmin232, M. Shimojima133, S. Shirabe100, M. Shiyakova95,ao, J. Shlomi228, A. Shmeleva127, D. Shoaleh Saadi126, M. J. Shochet47, S. Shojaii122, D. R. Shope145, S. Shrestha143, E. Shulga129, M. A. Shupe9, P. Sicho167, A. M. Sickles222, P. E. Sidebo198, E. Sideras Haddad195, O. Sidiropoulou230, D. Sidorov146, A. Sidoti27,28, F. Siegert68, Dj. Sijacki16, J. Silva160,163, S. B. Silverstein196, V. Simak168, Lj. Simic16, S. Simion149, 114 109 114 210 148 27 28 230 120 , , g , , p , , , , A. Shcherbakova196,197, C. Y. Shehu200, Y. Shen145, P. Sherwood109, L. Shi202,an, S. Shimizu97, C. O. Shimmin232, M. Shimojima133, S. Shirabe100, M. Shiyakova95,ao, J. Shlomi228, A. Shmeleva127, D. Shoaleh Saadi126, M. J. Shochet47, S. Shojaii122, D. R. Shope145, S. Shrestha143, E. Shulga129, M. A. Shupe9, P. Sicho167, A. M. Sickles222, P. E. Sidebo198, E. Sideras Haddad195, O. Sidiropoulou230, , y , , , , , j , p , S. Shrestha143, E. Shulga129, M. A. Shupe9, P. Sicho167, A. M. Sickles222, P. E. Sidebo198, E. Sideras Haddad195, O. Sidiropoulou230, E. Simioni114, B. Simmons109, M. Simon114, P. Sinervo210, N. B. Sinev148, M. Sioli27,28, G. Siragusa230, E. Simioni114, B. Simmons109, M. Simon114, P. Sinervo210, N. B. Sinev148, M. Sioli27,28, G. Siragusa230, I. Siral120, g S. Yu. Sivoklokov130, J. Sjölin196,197, M. B. Skinner102, P. Skubic145, M. Slater21, T. Slavicek168, M. Slaw j Smakhtin228, B. H. Smart7, J. 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Tsiskaridze72, Primary afliations 1 Primary afliations 1Department of Physics, University of Adelaide, Adelaide, Australia. 2Physics Department, SUNY Albany, Albany, New York, USA. 3Department of Physics, University of Alberta, Edmonton, Alberta, Canada. 4Department of Physics, Ankara University, Ankara, Turkey. 5Istanbul Aydin University, Istanbul, Turkey. 6Division of Physics, TOBB University of Economics and Technology, Ankara, Turkey. 7LAPP, CNRS/IN2P3 and Université Savoie Mont Blanc, Annecy-le-Vieux, France. 8High Energy Physics Division, Argonne National Laboratory, Argonne, Illinois, USA. 9Department of Physics, University of Arizona, Tucson, Arizona, USA. 10Department of Physics, The University of Texas at Arlington, Arlington, Texas, USA. 11Physics Department, National and Kapodistrian University of Athens, Athens, Greece. 12Physics Department, National Technical University of Athens, Zografou, Greece. 13Department of Physics, The University of Texas at Austin, Austin, Texas, USA. 14Institute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. 15Institut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain. 16Institute of Physics, University of Belgrade, Belgrade, Serbia. 17Department for Physics and Technology, University of Bergen, Bergen, Norway. 18Physics Division, Lawrence Berkeley National Laboratory and University of California, Berkeley, California, USA. 19Department of Physics, Humboldt University, Berlin, Germany. 20Albert Einstein Center for Fundamental Physics and Laboratory for High Energy Physics, University of Bern, Bern, Switzerland. 21School of Physics and Astronomy, University of Birmingham, Birmingham, UK. 22Department of Physics, Bogazici University, Istanbul, Turkey. 23Department of Physics Engineering, Gaziantep University, Gaziantep, Turkey. 24Istanbul Bilgi University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey. 25Bahcesehir University, Faculty of Engineering and Natural Sciences, Istanbul, Turkey. 26Centro de Investigaciones, Universidad Antonio Narino, Bogota, Colombia. 27INFN Sezione di Bologna, Bologna, Italy. 28Dipartimento di Fisica e Astronomia, Università di Bologna, Bologna, Italy. 29Physikalisches Institut, University of Bonn, Bonn, Germany. 30Department of Physics, Boston University, Boston, Massachusetts, USA. 31Department of Physics, Brandeis University, Waltham, Massachusetts, USA. 32Universidade Federal do Rio De Janeiro COPPE/EE/IF, Rio de Janeiro, Brazil. 33Electrical Circuits Department, Federal University of Juiz de Fora (UFJF), Juiz de Fora, Brazil. 34Federal University of Sao Joao del Rei (UFSJ), Sao Joao del Rei, Brazil. 35Instituto de Fisica, Universidade de Sao Paulo, Sao Paulo, Brazil. 36Physics Department, Brookhaven National Laboratory, Upton, New York, USA. 37Transilvania University of Brasov, Brasov, Romania. 38Horia Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania. 39Department of Physics, Alexandru Ioan Cuza University of Iasi, Iasi, Romania. 40National Institute for Research and Development of Isotopic and Molecular Technologies, Physics Department, Cluj Napoca, Romania. 41University Politehnica Bucharest, Bucharest, Romania. Primary afliations 1 42West University in Timisoara, Timisoara, Romania. 43Departamento de Física, Universidad de Buenos Aires, Buenos Aires, Argentina. 44Cavendish Laboratory, University of Cambridge, Cambridge, UK. 45Department of Physics, Carleton University, Ottawa, Ontario, Canada. 46CERN, Geneva, Switzerland. 47Enrico Fermi Institute, University of Chicago, Chicago, Illinois, USA. 48Departamento de Física, Pontiﬁcia Universidad Católica de Chile, Santiago, Chile. 49Departamento de Física, Universidad Técnica Federico Santa María, Valparaíso, Chile. 50Institute of High Energy Physics, Chinese Academy of Sciences, Beijing, China. 51Department of Physics, Nanjing University, Jiangsu, China. 52Physics Department, Tsinghua University, Beijing 100084, China. 53Department of Modern Physics, University of Science and Technology of China, Anhui, China. 54School of Physics, Shandong University, Shandong, China. 55Department of Physics and Astronomy, Key Laboratory for Particle Physics, Astrophysics and Cosmology, Ministry of Education, Shanghai Key Laboratory for Particle Physics and Cosmology, Shanghai Jiao Tong University, Shanghai (also at PKU-CHEP), China. 56Université Clermont Auvergne, CNRS/IN2P3, LPC, Clermont-Ferrand, France. 57Nevis Laboratory, Columbia University, Irvington, New York, USA. 58Niels Bohr Institute, University of Copenhagen, Kobenhavn, Denmark. 59INFN Gruppo Collegato di Cosenza, Laboratori Nazionali di Frascati, Rende, Italy. 60Dipartimento di Fisica, Università della Calabria, Rende, Italy. 61AGH University of Science and Technology, Faculty of Physics and Applied Computer Science, Krakow, Poland. 62Marian Smoluchowski Institute of Physics, Jagiellonian University, Krakow, Poland. 63Institute of Nuclear Physics Polish Academy of Sciences, Krakow, Poland. 64Physics Department, Southern Methodist University, Dallas, Texas, USA. 65Physics Department, University of Texas at Dallas, Richardson, Texas, USA. 66DESY, Hamburg and Zeuthen, Germany. 67Lehrstuhl für Experimentelle Physik IV, Technische Universität Dortmund, Dortmund, Germany. 68Institut für Kern- und Teilchenphysik, Technische Universität Dresden, Dresden, Germany. 69Department of Physics, Duke University, Durham, North Carolina, USA. 70SUPA - School of Physics and Astronomy, University of Edinburgh, Edinburgh, UK. 71INFN Laboratori Nazionali di Frascati, Frascati, Italy. 72Fakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany. 73Departement de Physique Nucleaire et Corpusculaire, Université de Genève, Geneva, Switzerland. 74INFN Sezione di Genova, Genova, Italy. 75Dipartimento di Fisica, Università di Genova, Genova, Italy. 76E. Andronikashvili Institute of Physics, Iv. Javakhishvili Tbilisi State University, Tbilisi, Georgia. 77High Energy Physics Institute, Tbilisi State University, Tbilisi, Georgia. 78II Physikalisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany. 79SUPA - School of Physics and Astronomy, University of Glasgow, Glasgow, UK. 80II Physikalisches Institut, Georg-August-Universität, Göttingen, Germany. 81Laboratoire de Physique Subatomique et de Cosmologie, Université Grenoble-Alpes, CNRS/IN2P3, Grenoble, France. 82Laboratory for Particle Physics and Cosmology, Harvard University, Cambridge, Massachusetts, USA. 83Kirchhof-Institut für Physik, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany. ARTICLES ARTICLES NATURE PHYSICS \| www.nature.com/naturephysics M. D. Werner94, P. Werner46, M. Wessels83, K. Whalen148, N. L. Whallon185, A. M. Wharton102, A. White10, M. J. White1, R. White49, D. Whiteson217, F. J. Wickens171, W. Wiedenmann229, M. Wielers171, C. 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Yuen29, I. Yusuf44,ax, B. Zabinski63, G. Zacharis12, R. Zaidan15, A. M. Zaitsev170,aj, N. Zakharchuk66, J. Zalieckas17, A. Zaman199, S. Zambito82, D. Zanzi119, C. Zeitnitz231, M. Zeman168, A. Zemla61, J. C. Zeng222, Q. Zeng190, O. Zenin170, T. Ženiš191, D. Zerwas149, D. Zhang120, F. Zhang229, G. Zhang53,aq, H. Zhang51, J. Zhang8, L. Zhang72, L. Zhang53, M. Zhang222, R. Zhang29, R. Zhang53,av, X. Zhang54, Y. Zhang50, Z. Zhang149, X. Zhao64, Y. Zhao54,ay, Z. Zhao53, A. Zhemchugov95, J. Zhong152, B. Zhou120, C. Zhou229, L. Zhou64, M. Zhou50, M. Zhou199, N. Zhou52, C. G. Zhu54, H. Zhu50, J. Zhu120, Y. Zhu53, X. Zhuang50, K. Zhukov127, A. Zibell230, D. Zieminska91, N. I. Zimine95, C. Zimmermann114, S. Zimmermann72, Z. Zinonos132, M. Zinser114, M. Ziolkowski188, L. Živković16, G. Zobernig229, A. Zoccoli27,28, R. Zou47, M. zur Nedden19, L. Zwalinski46 NATURE PHYSICS DOI: 10.1038/NPHYS4208 M. D. Werner94, P. Werner46, M. Wessels83, K. Whalen148, N. L. Whallon185, A. M. Wharton102, A. White10, M. J. White1, R. White49, D. Whiteson217, F. J. Wickens171, W. Wiedenmann229, M. Wielers171, C. Wiglesworth58, L. A. M. Wiik-Fuchs29, A. Wildauer132, F. Wilk115, H. G. Wilkens46, H. H. Williams155, S. Williams139, C. Willis121, S. Willocq117, J. A. Wilson21, I. Wingerter-Seez7, F. Winklmeier148, O. J. Winston200, B. T. Winter29, M. Wittgen190, M. Wobisch110,u, T. M. H. Wolf139, R. Wolf116, M. W. Wolter63, H. Wolters160,162, S. D. Worm21, B. K. Wosiek63, J. Wotschack46, M. J. Woudstra115, K. W. Wozniak63, M. Wu47, S. L. Wu229, X. Wu73, Y. Wu120, T. R. Wyatt115, B. M. Wynne70, S. Xella58, Z. Xi120, L. Xia52, D. Xu50 L. Xu36, B. Yabsley201, S. Yacoob193, D. Yamaguchi208, Y. Yamaguchi150, A. Yamamoto96, S. Yamamoto206, T. Yamanaka206, K. Yamauchi134, Y. Yamazaki97, Z. Yan30, H. Yang55, H. Yang18, Y. Yang202, Z. Yang17, W.-M. Yao18, Y. C. Yap111, Y. Yasu96, E. Yatsenko7, K. H. Yau Wong29, J. Ye64, S. Ye36, I. Yeletskikh95, E. Yigitbasi30, E. Yildirim114, K. Yorita227, K. Yoshihara155, C. Young190, C. J. S. Young46, S. Youssef30, D. R. Yu18, J. Yu10, J. Yu94, L. Yuan97, S. P. Y. Yuen29, I. Yusuf44,ax, B. Zabinski63, G. Zacharis12, R. Zaidan15, A. M. Zaitsev170,aj, N. Zakharchuk66, J. Zalieckas17, A. Zaman199, S. Zambito82, D. Zanzi119, C. Zeitnitz231, M. Zeman168, A. Zemla61, J. C. Zeng222, Q. Zeng190, O. Zenin170, T. Ženiš191, D. Zerwas149, D. Zhang120, F. Zhang229, G. Zhang53,aq, H. Zhang51, J. Zhang8, L. Zhang72, L. Zhang53, M. Zhang222, R. Zhang29, R. Zhang53,av, X. Zhang54, Y. Zhang50, Z. Zhang149, X. Zhao64, Y. Zhao54,ay, Z. Zhao53, A. Zhemchugov95, J. Zhong152, B. Zhou120, C. Zhou229, L. Zhou64, M. Zhou50, M. Zhou199, N. Zhou52, C. G. Zhu54, H. Zhu50, J. Zhu120, Y. Zhu53, X. Zhuang50, K. Zhukov127, A. Zibell230, D. Zieminska91, N. I. Zimine95, C. Zimmermann114, S. Zimmermann72, Z. Zinonos132, M. Zinser114, M. Ziolkowski188, L. Živković16, G. Zobernig229, A. Zoccoli27,28, R. Zou47, M. zur Nedden19, L. Zwalinski46 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS DOI: 10.1038/NPHYS4208 Smirn , , , , , , , , O. Smirnova112, J. W. Smith80, M. N. K. Smith57, R. W. Smith57, M. Smizanska102, K. Smolek168, A. A. Snesarev127, O. Smirnova112, J. W. Smith80, M. N. K. Smith57, R. W. Smith57, M. Smizanska102, K. Smolek168, A. , , , , , , I. M. Snyder148, S. Snyder36, R. Sobie225,o, F. Socher68, A. Sofer204, D. A. Soh202, G. Sokhrannyi106, M. Solar168, E. Yu. Soldatov129, U. Soldevila223, A. A. Solodkov170, A. Soloshenko95, O. V. Solovyanov170, V. Solovyev156, g p p A. M. Soukharev141,c, D. South66, B. C. Sowden108, S. Spagnolo103,104, M. Spalla157,158, M. Spangenberg226, F. Spanò108, p p p p g p g p p R. Stamen83, S. Stamm19, E. Stanecka63, R. W. Stanek8, C. Stanescu176, M. M. Stanitzki66, S. Stapnes151, E. A. Starchenko170, g H. J. Stelzer46, O. Stelzer-Chilton213, H. Stenzel78, G. A. Stewart79, J. A. Stillings29, M. C. Stockton148, M. Stoebe118, S. Strandberg196,197, A. Strandlie151, M. Strauss145, P. Strizenec192, R. Ströhmer230, D. M. Strom148, R. Stroynowski64, M. Swiatlowski47, S. P. Swift2, A. Sydorenko114, I. Sykora191, T. Sykora169, D. Ta72, K. Tackmann66, J. Taenzer204, A. Tafard217, J. Tanaka206, M. Tanaka208, R. Tanaka149, S. Tanaka96, R. Tanioka97, B. B. Tannenwald143, S. Tapia Araya49, S. Tapprogge114, A. C. Taylor137, G. N. Taylor119, P. T. E. Taylor119, W. Taylor214, P. Teixeira-Dias108, D. Temple189, H. Ten Kate46, P. K. Teng202, p J. P. Thomas21, J. Thomas-Wilsker108, P. D. Thompson21, A. S. Thompson79, L. A. Thomsen232, E. Thomson155, M. J. Tibbetts18, p S. Todorova-Nova7, J. Tojo100, S. Tokár191, K. Tokushuku96, E. Tolley82, L. Tomlinson115, M. Tomoto134, L. Tompkins190,as, C. J. Treado142, T. Trefzger230, A. Tricoli36, I. M. Trigger213, S. Trincaz-Duvoid111, M. F. Tripiana15, W A. Trofymov66, C. Troncon122, M. Trottier-McDonald18, M. Trovatelli225, L. Truong218,219, M. Trzebi y g K. W. Tsang87, J. C.-L. Tseng152, P. V. Tsiareshka124, G. Tsipolitis12, N. Tsirintanis11, S. Tsiskaridze15, E. G. Tskhadadze76, K. M. Tsui87, I. I. Tsukerman128, V. Tsulaia18, S. Tsuno96, D. Tsybychev199, Y. Tu88, A. Tudorache38, NATURE PHYSICS \| www.nature.com/naturephysics © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. NATURE PHYSICS \| www.nature.com/naturephysics ARTICLES ARTICLES © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Primary afliations 1 84Physikalisches Institut, Ruprecht-Karls-Universität Heidelberg, Heidelberg, Germany. 85ZITI Institut für Technische Informatik, Ruprecht-Karls-Universität Heidelberg, Mannheim, Germany. 86Faculty of Applied Information Science, Hiroshima Institute of Technology, Hiroshima, Japan. 87Department of Physics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong, China. 88Department of Physics, The University of Hong Kong, Hong Kong, China. NATURE PHYSICS DOI: 10.1038/NPHYS4208 NATURE PHYSICS DOI: 10.1038/NPHYS4208 NATURE PHYSICS DOI: 10.1038/NPHYS4208 89Department of Physics and Institute for Advanced Study, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. 90Department of Physics, National Tsing Hua University, Taiwan, Taiwan. 91Department of Physics, Indiana University, Bloomington, Indiana, USA. 92Institut für Astro- und Teilchenphysik, Leopold-Franzens-Universität, Innsbruck, Austria. 93University of Iowa, Iowa City, Iowa, USA. 94Department of Physics and Astronomy, Iowa State University, Ames, Iowa, USA. 95Joint Institute for Nuclear Research, JINR Dubna, Dubna, Russia. 96KEK, High Energy Accelerator Research Organization, Tsukuba, Japan. 97Graduate School of Science, Kobe University, Kobe, Japan. 98Faculty of Science, Kyoto University, Kyoto, Japan. 99Kyoto University of Education, Kyoto, Japan. 100Department of Physics, Kyushu University, Fukuoka, Japan. 101Instituto de Física La Plata, Universidad Nacional de La Plata and CONICET, La Plata, Argentina. 102Physics Department, Lancaster University, Lancaster, UK. 103INFN Sezione di Lecce, Lecce, Italy. 104Dipartimento di Matematica e Fisica, Università del Salento, Lecce, Italy. 105Oliver Lodge Laboratory, University of Liverpool, Liverpool, UK. 106Department of Experimental Particle Physics, Jožef Stefan Institute and Department of Physics, University of Ljubljana, Ljubljana, Slovenia. 107School of Physics and Astronomy, Queen Mary University of London, London, UK. 108Department of Physics, Royal Holloway University of London, Egham, UK. 109Department of Physics and Astronomy, University College London, London, UK. 110Louisiana Tech University, Ruston, Louisiana, USA. 111Laboratoire de Physique Nucléaire et de Hautes Energies, UPMC and Université Paris-Diderot and CNRS/IN2P3, Paris, France. 112Fysiska Institutionen, Lunds Universitet, Lund, Sweden. 113Departamento de Fisica Teorica C-15, Universidad Autonoma de Madrid, Madrid, Spain. 114Institut für Physik, Universität Mainz, Mainz, Germany. 115School of Physics and Astronomy, University of Manchester, Manchester, UK. 116CPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. 117Department of Physics, University of Massachusetts, Amherst, Massachusetts, USA. 118Department of Physics, McGill University, Montreal, Quebec, Canada. 119School of Physics, University of Melbourne, Victoria, Australia. 120Department of Physics, The University of Michigan, Ann Arbor, Michigan, USA. 121Department of Physics and Astronomy, Michigan State University, East Lansing, Michigan, USA. 122INFN Sezione di Milano, Milano, Italy. 123Dipartimento di Fisica, Università di Milano, Milano, Italy. 124B.I. Stepanov Institute of Physics, National Academy of Sciences of Belarus, Minsk, Republic of Belarus. 125Research Institute for Nuclear Problems of Byelorussian State University, Minsk, Republic of Belarus. 126Group of Particle Physics, University of Montreal, Montreal, Quebec, Canada. 127P.N. Lebedev Physical Institute of the Russian Academy of Sciences, Moscow, Russia. 128Institute for Theoretical and Experimental Physics (ITEP), Moscow, Russia. NATURE PHYSICS DOI: 10.1038/NPHYS4208 129National Research Nuclear University MEPhI, Moscow, Russia. 130D.V. Skobeltsyn Institute of Nuclear Physics, M.V. Lomonosov Moscow State University, Moscow, Russia. 131Fakultät für Physik, Ludwig-Maximilians-Universität München, München, Germany. 132Max-Planck-Institut für Physik (Werner-Heisenberg-Institut), München, Germany. 133Nagasaki Institute of Applied Science, Nagasaki, Japan. 134Graduate School of Science and Kobayashi-Maskawa Institute, Nagoya University, Nagoya, Japan. 135INFN Sezione di Napoli, Napoli, Italy. 136Dipartimento di Fisica, Università di Napoli, Napoli, Italy. 137Department of Physics and Astronomy, University of New Mexico, Albuquerque, New Mexico, USA. 138Institute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, the Netherlands. 139Nikhef National Institute for Subatomic Physics and University of Amsterdam, Amsterdam, the Netherlands. 140Department of Physics, Northern Illinois University, DeKalb, Illinois, USA. 141Budker Institute of Nuclear Physics, SB RAS, Novosibirsk, Russia. 142Department of Physics, New York University, New York, New York, USA. 143Ohio State University, Columbus, Ohio, USA. 144Faculty of Science, Okayama University, Okayama, Japan. 145Homer L. Dodge Department of Physics and Astronomy, University of Oklahoma, Norman, Oklahoma, USA. 146Department of Physics, Oklahoma State University, Stillwater, Oklahoma, USA. 147Palacký University, RCPTM, Olomouc, Czech Republic. 148Center for High Energy Physics, University of Oregon, Eugene, Oregon, USA. 149LAL, Univ. Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France. 150Graduate School of Science, Osaka University, Osaka, Japan. 151Department of Physics, University of Oslo, Oslo, Norway. 152Department of Physics, Oxford University, Oxford, UK. 153INFN Sezione di Pavia, Pavia, Italy. 154Dipartimento di Fisica, Università di Pavia, Pavia, Italy. 155Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania, USA. 156National Research Centre “Kurchatov Institute” B.P. Konstantinov Petersburg Nuclear Physics Institute, St Petersburg, Russia. 157INFN Sezione di Pisa, Pisa, Italy. 158Dipartimento di Fisica E. Fermi, Università di Pisa, Pisa, Italy. 159Department of Physics and Astronomy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 160Laboratório de Instrumentação e Física Experimental de Partículas - LIP, Lisboa, Portugal. 161Faculdade de Ciências, Universidade de Lisboa, Lisboa, Portugal. 162Department of Physics, University of Coimbra, Coimbra, Portugal. 163Centro de Física Nuclear da Universidade de Lisboa, Lisboa, Portugal. 164Departamento de Fisica, Universidade do Minho, Braga, Portugal. 165Departamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada (Spain), Portugal. 166Dep Fisica and CEFITEC of Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, Caparica, Portugal. 167Institute of Physics, Academy of Sciences of the Czech Republic, Praha, Czech Republic. 168Czech Technical University in Prague, Praha, Czech Republic. 169Charles University, Faculty of Mathematics and Physics, Prague, Czech Republic. NATURE PHYSICS DOI: 10.1038/NPHYS4208 170State Research Center Institute for High Energy Physics (Protvino), NRC KI, Russia. 171Particle Physics Department, Rutherford Appleton Laboratory, Didcot, UK. 172INFN Sezione di Roma, Roma, Italy. 173Dipartimento di Fisica, Sapienza Università di Roma, Roma, Italy. 174INFN Sezione di Roma Tor Vergata, Roma, Italy. 175Dipartimento di Fisica, Università di Roma Tor Vergata, Roma, Italy. 176INFN Sezione di Roma Tre, Roma, Italy. 177Dipartimento di Matematica e Fisica, Università Roma Tre, Roma, Italy. 178Faculté des Sciences Ain Chock, Réseau Universitaire de Physique des Hautes Energies - Université Hassan II, Casablanca, Morocco. 179Centre National de l’Energie des Sciences Techniques Nucleaires, Rabat, Morocco. 180Faculté des Sciences Semlalia, Université Cadi Ayyad, LPHEA-Marrakech, Morocco. 181Faculté des Sciences, Université Mohamed Premier and LPTPM, Oujda, Morocco. 182Faculté des sciences, Université Mohammed V, Rabat, Morocco. 183DSM/IRFU (Institut de Recherches sur les Lois Fondamentales de l’Univers), CEA Saclay (Commissariat à l’Energie Atomique et aux Energies Alternatives), Gif-sur-Yvette, France. 184Santa Cruz Institute for Particle Physics, University of California Santa Cruz, Santa Cruz, California, USA. 185Department of Physics, University of Washington, Seattle, Washington, USA. 186Department of Physics and Astronomy, University of Shefeld, Shefeld, UK. 187Department of Physics, Shinshu University, Nagano, Japan. 188Department Physik, Universität Siegen, Siegen, Germany. 189Department of Physics, Simon Fraser University, Burnaby, British Columbia, Canada. 190SLAC National Accelerator Laboratory, Stanford, California, USA. 191Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava, Slovak Republic. 192Department of Subnuclear Physics, Institute of Experimental Physics of the Slovak Academy of Sciences, Kosice, Slovak Republic. 193Department of Physics, University of Cape Town, Cape Town, South Africa. 194Department of Physics, University of Johannesburg, Johannesburg, South Africa. 195School of Physics, University of the Witwatersrand, Johannesburg, South Africa. 196Department of Physics, Stockholm University, Stockholm, Sweden. 197The Oskar Klein Centre, Stockholm, Sweden. 198Physics Department, Royal Institute of Technology, Stockholm, Sweden. 199Departments of Physics & Astronomy and Chemistry, Stony Brook University, Stony Brook, New York, USA. 200Department of Physics and Astronomy, University of Sussex, Brighton, UK. 201School of Physics, University of Sydney, Sydney, Australia. 202Institute of Physics, Academia Sinica, Taipei, Taiwan. 203Department of Physics, Technion: Israel Institute of Technology, Haifa, Israel. 204Raymond and Beverly Sackler School of Physics and Astronomy, Tel Aviv University, Tel Aviv, Israel. 205Department of Physics, Aristotle University of Thessaloniki, Thessaloniki, Greece. 206International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan. 207Graduate School of Science and Technology, Tokyo Metropolitan University, Tokyo, Japan. 208Department of Physics, Tokyo Institute of Technology, Tokyo, Japan. Secondary afliations y aDepartment of Physics, King’s College London, London, UK. bInstitute of Physics, Azerbaijan Academy of Sciences, Baku, Azerbaijan. cNovosibirsk State University, Novosibirsk, Russia. dTRIUMF, Vancouver, British Columbia, Canada. eDepartment of Physics & Astronomy, University of Louisville, Louisville, Kentucky, USA. fPhysics Department, An-Najah National University, Nablus, Palestine. gDepartment of Physics, California State University, Fresno, California, USA. hDepartment of Physics, University of Fribourg, Fribourg, Switzerland. iII Physikalisches Institut, Georg-August-Universität, Göttingen, Germany. jDepartament de Fisica de la Universitat Autonoma de Barcelona, Barcelona, Spain. kDepartamento de Fisica e Astronomia, Faculdade de Ciencias, Universidade do Porto, Portugal. lTomsk State University, Tomsk, Russia. mThe Collaborative Innovation Center of Quantum Matter (CICQM), Beijing, China. nUniversita di Napoli Parthenope, Napoli, Italy. oInstitute of Particle Physics (IPP), Canada. pHoria Hulubei National Institute of Physics and Nuclear Engineering, Bucharest, Romania. qDepartment of Physics, St Petersburg State Polytechnical University, St Petersburg, Russia. rBorough of Manhattan Community College, City University of New York, New York City, USA. sDepartment of Physics, The University of Michigan, Ann Arbor, Michigan, USA. tCentre for High Performance Computing, CSIR Campus, Rosebank, Cape Town, South Africa. uLouisiana Tech University, Ruston, Louisiana, USA. vInstitucio Catalana de Recerca i Estudis Avancats, ICREA, Barcelona, Spain. wGraduate School of Science, Osaka University, Osaka, Japan. xFakultät für Mathematik und Physik, Albert-Ludwigs-Universität, Freiburg, Germany. yInstitute for Mathematics, Astrophysics and Particle Physics, Radboud University Nijmegen/Nikhef, Nijmegen, the Netherlands. zDepartment of Physics, The University of Texas at Austin, Austin, Texas, USA. aaInstitute of Theoretical Physics, Ilia State University, Tbilisi, Georgia. abCERN, Geneva, Switzerland. acGeorgian Technical University (GTU), Tbilisi, Georgia. adOchadai Academic Production, Ochanomizu University, Tokyo, Japan. aeManhattan College, New York, New York, USA. afAcademia Sinica Grid Computing, Institute of Physics, Academia Sinica, Taipei, Taiwan. agSchool of Physics, Shandong University, Shandong, China. ahDepartamento de Fisica Teorica y del Cosmos and CAFPE, Universidad de Granada, Granada (Spain), Portugal. aiDepartment of Physics, California State University, Sacramento, California, USA. ajMoscow Institute of Physics and Technology State University, Dolgoprudny, Russia. akDepartement de Physique Nucleaire et Corpusculaire, Université de Genève, Geneva, Switzerland. alInternational School for Advanced Studies (SISSA), Trieste, Italy. amInstitut de Física d’Altes Energies (IFAE), The Barcelona Institute of Science and Technology, Barcelona, Spain. anSchool of Physics, Sun Yat-sen University, Guangzhou, China. aoInstitute for Nuclear Research and Nuclear Energy (INRNE) of the Bulgarian Academy of Sciences, Soﬁa, Bulgaria. apFaculty of Physics, M.V. Lomonosov Moscow State University, Moscow, Russia. aqInstitute of Physics, Academia Sinica, Taipei, Taiwan. NATURE PHYSICS DOI: 10.1038/NPHYS4208 Physics and Astronomy, University of Victoria, Victoria, British Columbia, Canada. 226Department of Physics, University of Warwick, Coventry, UK. 227Waseda University, Tokyo, Japan. 228Department of Particle Physics, The Weizmann Institute of Science, Rehovot, Israel. 229Department of Physics, University of Wisconsin, Madison, Wisconsin, USA. 230Fakultät für Physik und Astronomie, Julius-Maximilians-Universität, Würzburg, Germany. 231Fakultät für Mathematik und Naturwissenschaften, Fachgruppe Physik, Bergische Universität Wuppertal, Wuppertal, Germany. 232Department of Physics, Yale University, New Haven, Connecticut, USA. 233Yerevan Physics Institute, Yerevan, Armenia. 234CH-1211, Geneva 23, Switzerland. 235Centre de Calcul de l’Institut National de Physique Nucléaire et de Physique des Particules (IN2P3), Villeurbanne, France. NATURE PHYSICS DOI: 10.1038/NPHYS4208 209Tomsk State University, Tomsk, Russia. 210Department of Physics, University of Toronto, Toronto, Ontario, Canada. 211INFN-TIFPA, Trento, Italy. 212University of Trento, Trento, Italy. 213TRIUMF, Vancouver, British Columbia, Canada. 214Department of Physics and Astronomy, York University, Toronto, Ontario, Canada. 215Faculty of Pure and Applied Sciences, and Center for Integrated Research in Fundamental Science and Engineering, University of Tsukuba Tsukuba Japan 216Department of Physics and Astronomy Tufts University Medford Massachusetts USA 217Department of Physics and NATURE PHYSICS \| www.nature.com/naturephysics ARTICLES NATURE PHYSICS \| www.nature.com/naturephysics © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. Secondary afliations arNational Research Nuclear University MEPhI, Moscow, Russia. asDepartment of Physics, Stanford University, Stanford, California, USA. atInstitute for Particle and Nuclear Physics, Wigner Research Centre for Physics, Budapest, Hungary. auGiresun University, Faculty of Engineering, Turkey. avCPPM, Aix-Marseille Université and CNRS/IN2P3, Marseille, France. awDepartment of Physics, Nanjing University, Jiangsu, China. axUniversity of Malaya, Department of Physics, Kuala Lumpur, Malaysia. ayLAL, Univ. Paris-Sud, CNRS/IN2P3, Université Paris-Saclay, Orsay, France. ‡Deceased. *e-mail: atlas.publications@cern.ch NATURE PHYSICS \| www.nature.com/naturephysics
https://openalex.org/W4384108521	https://hal.science/hal-04230155/file/How%20Can%20I%20Help%20You.pdf	English	null	How Can I Help You? The Influence of Situation and Hostile Sexism on Perception of Appropriate Gender of Conversational Agents	Revue internationale de psychologie sociale	2,023	cc-by	12,501	To cite this version: Mathieu Pinelli, Elisa Sarda, Clémentine Bry. How Can I Help You? The Influence of Situation and Hostile Sexism on Perception of Appropriate Gender of Conversational Agents. International Review of Social Psychology, 2023, 36 (1), pp.10. 10.5334/irsp.669. hal-04230155 Distributed under a Creative Commons Attribution 4.0 International License MATHIEU PINELLI ELISA SARDA CLÉMENTINE BRY Author affiliations can be found in the back matter of this article Mathieu Pinelli Conversational agents (CAs) are increasingly being developed on commercial websites nowadays. We tested in two studies whether gender stereotypes apply to non-gendered CAs. In the first study, participants evaluated whether CAs are expected to display more masculine or feminine characteristics in situations designed to be stereotypically male or female. The sexist attitudes of the respondents were also measured. As predicted, participants perceived that a CA should be more masculine in stereotypically male situations and more feminine in stereotypically female situations. Moreover, we found that hostile sexism but not benevolent sexism moderated the effect of the gendered situation. The second study replicated the results while addressing the limits of Study 1, showing the robustness of these effects. These findings are consistent with models of gender stereotypes in humans and robots and show for the first time a moderation effect of (hostile) sexism in a customer service context with CAs. The processes involved in human relationships seem relevant in a digital environment that involves CAs. Researchers and professionals should work together to avoid reproducing and perpetuating gender stereotypes when developing CAs. Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, LPNC, 38000 Grenoble, France Université Grenoble Alpes, Université Savoie Mont Blanc, CNRS, LPNC, 38000 Grenoble, France mathieu.pinelli@univ- grenoble-alpes.fr mathieu.pinelli@univ- grenoble-alpes.fr KEYWORDS: Ambivalent Sexism; Gender Biases; Conversational Agents HAL Id: hal-04230155 https://hal.science/hal-04230155v1 Submitted on 5 Oct 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License RESEARCH ARTICLE How Can I Help You? The Influence of Situation and Hostile Sexism on Perception of Appropriate Gender of Conversational Agents MATHIEU PINELLI ELISA SARDA CLÉMENTINE BRY Author affiliations can be found in the back matter of this article How Can I Help You? The Influence of Situation and Hostile Sexism on Perception of Appropriate Gender of Conversational Agents RESEARCH ARTICLE MATHIEU PINELLI ELISA SARDA CLÉMENTINE BRY INTRODUCTION Situations involving care and communality are deemed more appropriate for women, and reciprocally women are perceived as better suited for care and warmth situations. On the other hand, situations that require competence, assertiveness, and confidence are deemed more appropriate for men, and reciprocally men are perceived as better suited for competence and assertiveness situations (Eagly & Wood, 2012; Ellemers, 2018). Robots and artificial intelligence applications are increasingly being used on-line to help users with customer services and to simulate a realistic human presence. We focus in this paper on conversational agents (CAs) designed to interact with humans using natural language (Dale, 2016; Feine et al., 2019). Conversational agents are almost a must-have on a commercial website these days (e.g., there were 300,000 CAs on Facebook in 2018),1 and they have positive consequences on users by increasing satisfaction and giving the feeling of a social presence (Chung et al., 2020; Feine et al., 2019). Conversational agents can be found in the form of personal assistants (e.g., Cortana, Alexa, Siri), as customer services support, in multiple technical support roles (smartphones, tablets, or computers), and in various fields, such as education, healthcare, and marketing (Bickmore & Gruber, 2010; Chung et al., 2020; Provoost et al., 2017; Tegos & Demetriadis, 2017). Conversational agents are increasingly sophisticated and are used on a daily basis in direct contact with users in a B2C context (e.g., Chung et al., 2020). The development of CAs requires trade-offs between different technical and social features (Feine et al., 2019). One of the inevitable questions lies in relation to a possible gender for CAs, as CAs are used to increase the feeling of a human social presence and human interactions are coloured, for better or for worse, by gender and gendered behavioural expectations. Users may therefore expect gendered features for CAs; at least, developers seem to think so and have therefore produced gendered CAs.2 In this paper, we question whether people actually expect a gendered CA and the factors that would trigger such gendered expectations. No experimental study to our knowledge has studied the gendered expectations in relation to CAs before. The literature about gender features in human-human interactions and in robot- human interactions can help delineate what we can expect from CAs. From the gender stereotype literature, we can infer that some people could expect an interaction agent (here a CA) to match a specific gender social role. INTRODUCTION Ellemers, 2018; Spencer et al., 2016), thus reinforcing themselves as men and women adopt gendered social roles (Eagly & Wood, 2012). These gendered social roles give the impression that they are innate and inevitable, and therefore seem to be inherent in our society (Eagly & Wood, 2016). Interactions between machines and humans have aroused many fantasies since the early development of computers, robots, and artificial intelligence. The claims that ‘Machines will replace humans’ or ‘we will no longer differentiate between humans and machines’ are often heard in everyday talk. Fiction stories about machines taking over humans are numerous (e.g., The Terminator, The Matrix, and Westworld, to name just a few films and TV shows). Interactions between machines and humans have aroused many fantasies since the early development of computers, robots, and artificial intelligence. The claims that ‘Machines will replace humans’ or ‘we will no longer differentiate between humans and machines’ are often heard in everyday talk. Fiction stories about machines taking over humans are numerous (e.g., The Terminator, The Matrix, and Westworld, to name just a few films and TV shows). Gender stereotypes are both descriptive (that is, what people are) and prescriptive (that is, what people should be; Prentice & Carranza, 2002; Eagly & Karau, 2002; Ellemers, 2018). Extensive research has identified two core dimensions in social perception: Communion and Agency (or warmth and competence; see, for instance, Abele et al., 2008; Fiske et al., 2007; Judd et al., 2005). Communion is related to warmth, sympathy, emotional sensitivity, and concern with others, whereas Agency is related to competence, assertiveness, confidence, and self-control (e.g., Cuddy et al., 2008; Eagly & Karau, 2002). Social perception research has found that men are described as more agentic than women and that women are described as more communal than men (Eagly & Steffen, 1984; Ellemers, 2018). Furthermore, matching the prescription, men’s behaviour is expected to be related to competence and agency, while women’s behaviour is expected to be related to warmth and care (Prentice & Carranza, 2002). These gender norms define what traits are acceptable (or unacceptable) for men and women, and breaking the gender norms can lead to prejudice (e.g., Eagly & Karau, 2002). Gender norms define the behaviour that women and men should display and, thus, the situations that conform to each gender. TO CITE THIS ARTICLE: Pinelli, M., Sarda, E., & Bry, C. (2023). How Can I Help You? The Influence of Situation and Hostile Sexism on Perception of Appropriate Gender of Conversational Agents. International Review of Social Psychology, 36(1): 10, 1–16. DOI: https://doi.org/10.5334/ irsp.669 Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 2 INTRODUCTION The gender role could be cued, for instance, by the situation at hand. A situation involving warmth and care would cue to a female gender role, while a situation involving competence and assertiveness would cue to a male gender role. Interestingly, CAs are used in a variety of situations, with some situations being more related to warmth and care (e.g., using the guarantee attached to a hairdressing appliance) and other situations being related to competence and assertiveness (e.g., financial services allowing customers to save and invest money). Users could expect the CA to conform to a female gender role in a warmth-related situation, whereas they may expect the CA to conform to a male gender role in a competence-related situation. At least, those predictions GENDER IN HUMAN-ROBOT INTERACTIONS Overall, the literature shows that people interact with CAs or robots in a similar way as they do with human fellows. Sometimes, these interactions with CAs or robots can also exacerbate negative social processes such as gender stereotypes, harassment, or gender-based division of labour with the consequence of reproducing and reinforcing sexism daily in our society (Brahnam & De Angeli, 2012; Eyssel & Hegel, 2012; Nomura & Suzuki, 2022). Some studies have shown that people react to computers in the same way as they do to humans (Feine et al., 2019; Nass & Moon, 2000), and that people are able to interact with computers in the same way as they do with humans (Nass et al., 1997). The Computers Are Social Actors (CASA) model states that people interacting with computers have social reactions similar to human social interactions according to social cues like voice, gesture, physical design, or the apparent ‘gender’ (e.g., Eyssel & Hegel, 2012; Feine et al., 2019; Gong, 2008; Nass et al., 1997). It appears that gender roles are used to interact with CAs and that gender stereotypes are applied to CAs as well as humans. Human features (e.g., a voice and/or a face) are implemented to improve the user’s experience, giving a personalized service anytime and anywhere (e.g., Chung et al., 2020), and these human features can increase inferences of social roles. However, with CAs, the interactions are generally in a written form, through a chat, which means that such human features are not relevant. There might sometimes be an avatar displaying a male or female character, but this gendered avatar is not systematically present. Therefore, most CAs could be more gender neutral than robots. Unable to rely on gendered features, will people still project gender roles on CAs? When the CA has no gender feature, is the (gendered) situation enough to trigger gender expectations toward the conversational agent? Actually, we believe that adherence to sexism could play a role. Voice is an important social cue defining personality and gender attribution. Nass et al. (1997) found that a high-pitched synthetic voice was associated with a ‘female’ computer, whereas a low-pitched synthetic voice was associated with a ‘male’ computer. Their study showed that humans react to a computer by applying the same social rules they usually reserve for social interactions between humans (see also: Nass & Moon, 2000). GENDER IN HUMAN INTERACTIONS Gender and its associated beliefs are central in our social relationships (Eagly & Wood, 2016; Ellemers, 2018). Men and women are believed to be similar in some ways but very different in many other ways. These beliefs influence not only our perceptions, but also our behaviour (e.g., Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 3 communication during interaction with CAs are common (De Angeli & Brahnam, 2008). For example, Brahnam and De Angeli (2012) showed that 18% of the conversation was focused on sexual attention and negative stereotypes with female CAs compared to 10% with male CAs and only 2% with non-gendered CAs. would hold if social roles were to be applied to artificial intelligence and machines. The literature on robot- human interaction may help us understand whether there is solid ground for such hypotheses. GENDER IN HUMAN-ROBOT INTERACTIONS More recently, Eyssel and Hegel (2012) tested the effect of gendered facial features of robots on perception and description. They reported that short-haired robots (i.e., those with a male facial feature) were perceived as more agentic than long-haired robots (i.e., those with a female facial feature), which were perceived as more communal. Furthermore, tasks (such as repairing technical equipment) were perceived as more suitable for a ‘male’ robot and conversely female-dominated tasks dominated by women (such as household maintenance) were perceived as more suitable for a ‘female’ robot. More recently, Bernotat et al. (2021) showed how body shape also influences the perception of a robot. Their results indicated that stereotypically female activities and communal attributions were associated with a robot with a female body shape rather than with a male body shape. Furthermore, they showed that benevolent sexism (but not hostile sexism) marginally affected the agency attribution. Correlation analysis showed that the higher benevolent sexism was, the more agency was attributed to the robot. SEXIST ATTITUDES Gender stereotypes have been extensively studied in human interactions, and some studies have extended that literature to robot interactions. In human interactions, the use of gender stereotypes depends on sexist attitudes. Sexism was once studied as a unitary dimension, but Glick and Fiske (1996) offered a more nuanced definition with their theory of ambivalent sexism. They proposed that two sorts of sexism coexist, as the two faces of the same coin: hostile sexism and benevolent sexism. Hostile sexism matches the more traditional sexist attitudes reviewed in the literature, comprising a negative attitude towards women, with feelings of antipathy and a fear that women will take power over men (Glick & Fiske, 1996). Hostile sexism can be expressed through discrimination in employment. Studies have shown, for example, that individuals higher in hostile sexism are less likely to recommend a female candidate for a managerial position (Masser & Abrams, 2004). Benevolent sexism, on the other hand, can be seen as a ‘more positive’ attitude toward women, associated with chivalry and paternalistic attitudes (Glick & Fiske, Therefore, gender stereotypes are applied to robots. Several studies have extended this research to CAs, showing that social features affect users’ satisfaction, but also their perceptions of truthfulness, credibility, and social presence (Araujo, 2018; McDonnell & Baxter, 2019; Toader et al., 2020; Verhagen et al., 2014). Humans can interact with CAs in a natural language and adopt behaviours they usually have with their peers, that includes abuse, harassment, and mistreatment (Brahnam & De Angeli, 2012). Verbal abuse and sexual Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 4 stereotypically male situations and the female gender to be more appropriate in stereotypically female situations. H1b: Participants would deem warmth features more appropriate for the CA in stereotypically female situations and competence features more appropriate in stereotypically male situations. H1c: The effect of stereotypically male and female situations would be moderated by sexist attitudes such that the more sexist (hostile and/or benevolent) the participant, is the more they would rely on gender stereotypes in their evaluation of the appropriate characteristics of the CAs. 1996). In this form of sexism, women are perceived as having a higher moral purity than men and as too fragile to undertake tasks involving strength (protective paternalism). They are also perceived as creatures without whom men cannot be complete and possess qualities that men do not possess. THE CURRENT RESEARCH A power analysis was performed using GPower 3.1 (Faul et al., 2007) with a small to moderate effect size of f² = .10, using a within-subjects design and based on the literature on sexism (e.g., McCarty & Kelly, 2015). This power analysis suggested that we needed 114 participants for a power level of .80. Thus, 117 participants took part in our online study. French-speaking participants were recruited on the Prolific platform (only participants with 95% positive rates were included) and they received £0.84 for their participation. Fifteen participants were excluded after an initial sort,3 so the final sample included 102 participants (Mage = 30.54, SD = 10.56; 38 women and 64 men). As we do not meet the number of participants recommended by the power analysis, we performed a sensitivity analysis to indicate what effect size was detectable with the final sample at 80% power (threshold of .05, 102 participants, and 20 predictors in the linear model) using GPower. The analysis indicated that with this design, the minimum effect we could detect would be f² = .11. Gender stereotypes infuse our social life and influence our interactions in a variety of contexts, including marketing, workplaces, and robot interactions (Bernotat et al., 2021; Grau & Zotos, 2016; Koch et al., 2015). With digital growth, the question of the influence of gender stereotypes in digital contexts involving virtual CAs is of importance. Several previous studies have focused on gender stereotypes in robots (e.g., Eyssel & Hegel, 2012), but no study has experimentally tested gender biases and sexist attitudes with CAs. We believe that there is little reason to expect that gendered CAs would not trigger gender stereotyping. However, we wondered whether neutral CAs would still be the target of sexist stereotypes and if stereotyping would be predicted by the participants’ own level of sexist attitude (i.e., hostile and benevolent sexism). We reasoned that according to the commercial service one is looking for (e.g., advice about saving money vs. finding beauty products), people could consider the situation as stereotypically masculine or feminine. Our two studies aimed to test the impact of stereotypically male and female situations on the perception of appropriate features for CAs (gender, warmth, and competence) and the moderator effect of ambivalent sexism, represented by hostile and benevolent sexism. SEXIST ATTITUDES Those individuals higher in benevolent sexism therefore assign women to less challenging tasks (King et al., 2012), and perceive men as more agentic and women as more communal (Rudman & Kilianski, 2000). Benevolent sexism can be seen as more positive than hostile sexism, though both attitudes involve prejudice against women, placing them below men (e.g., Stermer & Burkley, 2015). For example, by describing women as warmer than men, benevolent sexism suggests that women are less competent than men (Kervyn et al., 2012). stereotypically male situations and the female gender to be more appropriate in stereotypically female situations. H1b: Participants would deem warmth features more appropriate for the CA in stereotypically female situations and competence features more appropriate in stereotypically male situations. H1c: The effect of stereotypically male and female situations would be moderated by sexist attitudes such that the more sexist (hostile and/or benevolent) the participant, is the more they would rely on gender stereotypes in their evaluation of the appropriate characteristics of the CAs. Independent Variable with Random Effects The participants and the nine situations were variables with random effects. Therefore, we included in the model the estimation of their intercept and slope by situation or slope by sexism level, respectively.6 Independent Variable with Fixed Effects: The Situations We created two contrasts to test a linear trend from stereotypically male to the stereotypically female situations through the neutral one. We coded the first contrast C1: female = +1; neutral = 0; male = –1, and the residual contrast C2: female = –1; neutral = +2; male = –1. If the trend is linear, we expect that C1 is significant and C2 to be not significant. For each situation, the participants answered a questionnaire on the CA’s appropriate features. They first evaluated the appropriate CA gender (from 1 = male to 5 = female), and its appropriate age (in its twenties, thirties, forties, or fifties). Then participants were required to rate the relevance of eight traits for the CA on a Likert scale ranging from 1 (not at all) to 5 (very much). Agency and communion traits were used to study gender stereotypes in robots. However, agency is related to actions in the world, which is not relevant to conversation agents. We therefore chose traits related to competence and warmth instead, as they are more general (see Cuddy et al., 2008). These items were adapted from Fiske et al. (2002). Trustworthy, friendly, well-intentioned, and warm evaluated the warmth dimension, and competent, intelligent, capable, and efficient evaluated the competence dimension. Both contrasts C1 and C2, participants’ gender (–1 = woman, +1 = man), benevolent sexism (centred), hostile sexism (centred), suspicion level (centred), and attitude toward CAs (centred) were entered as fixed effects in the linear mixed-effect model (see Judd et al., 2012). All measures showed good internal consistency (see Table 1). Following Judd et al. (2012) and Judd et al. (2017), we compared models with and without each random parameter in order to retain the most conservative model. We followed the same rationale with fixed effects.7 The results corresponding to the tested hypotheses are presented in Table 1 (see mixed- effects models on our OSF page). Participants then completed a post-experimental questionnaire. We measured the attitude toward CAs with four items adapted from Venkatesh et al. (2003) on a 7-point Likert scale, and one item measured the frequency of use (from 1 = Never to 5 = Very often). Analysis Plan Given our design, we used linear mixed-effects models with fixed and random effects variables. All analyses were conducted in R, using mixed-effects models with the lme4 package (Bates et al., 2021). Mixed models allow the use of fixed-effect variables (as in ANOVA) and random-effect variables. The participants then moved on to the alleged Study 2, presented as a marketing research about the development of online CAs. A conversational agent was defined as ‘a computer program capable of conducting a conversation’, so that all participants had the same representation of a CA. The participants were told that they would be presented with different online situations in which a customer (of unspecified gender)4 would resort to a CA to answer their request. The participants’ task would be to indicate the CA’s most appropriate features to match the customer’s needs in each situation. Participants were instructed to answer from the customer’s point of view and not from their own, in order to limit social desirability bias (Fiske et al., 2002). Nine situations were presented in a random order to each participant (using a within-subjects design). The situations5 were related to online banking services and to retail websites, and were designed to conform to stereotypically male (N = 3), female (N = 3), or neutral (N = 3) gender norms. Material and Procedure To reduce participants’ suspicions towards the purpose and hypothesis of the study, the cover story presented the two parts as two separate studies, which were said to be combined for economic reasons. The alleged goal of the first ‘study’ was to validate questionnaires in different domains (marketing, ecology, gender perception). The participants were informed that they would randomly answer only one of three possible questionnaires. Actually, they always answered the gender perception questionnaire, which consisted of the Ambivalent Sexism Inventory (Glick & Fiske, 1996) validated in French (Dardenne et al., 2006). We used the short version of Rollero et al. (2014). The scale consists of two dimensions: hostile sexism and benevolent sexism. Both subscales are composed of six items (e.g., women seek power by having control over men; many women have a kind of purity that men do not). In this paper, we extend previous work and test whether perceptions of gender-undefined CAs are also influenced by gender stereotypes and sexist attitudes. In two studies, participants were presented with several stereotypically ‘gendered’ situations in which they had to indicate the most appropriate characteristics (i.e., gender, warmth, and competence traits) for the CA. We formulate the following hypothesis: H1a: Participants would consider the male gender to be more appropriate for the CA in Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 5 The participants provided a response for each item on a scale from 1 (not at all) to 6 (completely) and obtained a mean score for hostile sexism and a mean score for benevolent sexism. Dependent Variables We computed a perceived appropriate gender (1 = male to 5 = female), an appropriate warmth index (mean evaluation of warmth traits from 1 = not at all to 5 = very much), and an appropriate competence index (mean evaluation of competence traits from 1 = not at all to 5 = very much) for each scenario. Table 1 Means (SD) and Cronbach’s alpha of variables included in the model (Study 1). Independent Variable with Random Effects The five items comprised an attitude index (α = .88). The higher the score, the more positive is the participant’s attitude towards CAs. We checked for suspicions regarding the actual/alleged goals of the study and the possible influence between the different parts, with three open questions. The answers were coded by the authors and rated from 0 = not suspicious to 3 = completely suspicious. Finally, a socio-demographic questionnaire collected the age, sex, socio-professional category, and nationality of each participant. MEAN (SD) CRONBACH’S ALPHA Hostile sexism 2.58 (1.21) .90 Benevolent sexism 2.88 (1.11) .82 Appropriate Competence 4.50 (0.50) .73 Appropriate Warmth 4.07 (0.60) .74 Attitudes toward CAs 4.52 (1.14) .88 Table 1 Means (SD) and Cronbach’s alpha of variables included in the model (Study 1). Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 6 than in the male stereotypical situations, when they have a more positive attitude toward CAs (Figure 3). The Appropriate Gender of the Conversational Agent Suspicion level, gender of participants, attitudes towards CAs, and benevolent sexism did not have a valuable input in the model and were therefore discarded. We found a significant effect of C1, t = 3.12, p = .016, but not of C2, p = .90. As expected, we found a significant effect of stereotypical situations. We observed that the appropriate gender linearly increases toward femininity (Figure 1) when passing from stereotypically masculine situations (M = 2.67; SD = 0.76) to stereotypically feminine situations (M = 3.42; SD = 0.78). Discussion of Study 1 The goal of this first study was to test the effect of stereotypically gendered situations on the expected features of a neutral conversational agent, according to hostile and benevolent sexism. The results partly support our hypothesis. The appropriate gender for a neutral CA was regarded as more female in stereotypically female situations and more male in stereotypically male situations, and this effect increased according to the level of hostile sexism. In addition, the competence traits were perceived more appropriate in male situations than in female situations. Interestingly, we did not find these effects in relation to warmth. Instead, the appropriate level of warmth was predicted by participants’ attitudes towards CAs differently in stereotypically male and female situations. Specifically, the more positive the participant’s The Appropriate Level of Competence Suspicion level, gender of participants, attitudes towards CAs, hostile sexism and benevolent sexism were found to have no valuable input in the model, so these variables were discarded. We found a significant effect of C1 on the appropriate level of competence, t = –3.03, p = .017, but not of C2, p = .51. The results showed a linear decrease in the appropriate level of competence when moving from stereotypically masculine situations (M = 4.62; SD = 0.34) to stereotypically female situations (M = 4.46; SD = 0.40). Moreover, the interaction between hostile sexism and C1 was significant, t = 3.82, p = .002, and the interaction with C2 was not, p = .90. The effect of the stereotypical situations increases with participants’ hostile sexism. The more sexist the participants are, the more they consider that the CAs’ gender should match the gendered situations (see Figure 2). The Appropriate Level of Warmth The suspicion level, the gender of the participants, and hostile sexism did not have a valuable input into the model and therefore were discarded. We did not find a significant effect of gendered situations on the appropriate level of warmth, C1: t < 1, p = .61, C2, t < 1, p = .36. We did not find a significant interaction with benevolent sexism. The interaction between the attitude towards CAs and C1 was significant, t = 2.91, p = .003, but not with C2, p = .66. Participants perceived warmth to be more appropriate in the female stereotypical situations Figure 1 Effect of the stereotypical situations on the CA’s appropriate gender (bars represent confidence intervals). Figure 1 Effect of the stereotypical situations on the CA’s appropriate gender (bars represent confidence intervals Figure 1 Effect of the stereotypical situations on the CA’s appropriate gender (bars represent confidence intervals). Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 7 7 Figure 2 Conversational agents appropriate gender according to the gendered situations (represented by C1) and hostile sexism (centred). A lower value in the appropriate gender corresponds to a rather masculine gender, and a higher value corresponds to a rather feminine gender. Figure 2 Conversational agents appropriate gender according to the gendered situations (represented by C1) and hostile sexism (centred). A lower value in the appropriate gender corresponds to a rather masculine gender, and a higher value corresponds to a rather feminine gender. Figure 3 Appropriate level of warmth for the Conversational Agent according to the gendered situations (represented by C1) and attitude towards CAs (centred). Figure 3 Appropriate level of warmth for the Conversational Agent according to the gendered situations (represented by C1) and attitude towards CAs (centred). attitude toward CAs is, the more the participants perceived warmth as appropriate in stereotypically female situations compared to stereotypically male situations. This effect was not expected and needs replication. In this study we did not control the customer’s gender and used a within-subjects design: Participants were exposed to the nine situations. The within-subjects design may have increased the participants’ awareness of our hypotheses related to gender stereotypes. Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 8 evaluation of CAs, regardless of the customer gender. Furthermore, the customer’s gender being unspecified, the gendered situations may have influenced not only the CA’s perceptions but also the perceptions of the customer. Method Participants Three hundred and eighteen persons participated in this online study (the result of the power analysis was N = 245, which was carried out to detect main and interaction effects with an effect size of f = .20, a power of .80, and using a between-subjects design). French- speaking participants were recruited on the Prolific platform (participants with 95% of positive rates were included) and received £0.84 for their participation, similar to the first study. Forty-seven participants were discarded (based on the time taken to fill out the study and the distraction level). The final sample included 271 participants (Mage = 29.84, SD = 10.44; 113 women and 158 men). We performed a sensitivity analysis to indicate what effect size was detectable with the final sample (setting an error alpha rate of .05, a power of .80, 271 participants and 6 groups). The analysis indicated that with this design, the minimum effect we could detect would be a f = .18 (d = .36). Material and Procedure Similar to Study 1, Study 2 was presented as two supposedly separate studies. Participants completed the short version of the ambivalent sexism questionnaire (Rollero et al., 2014) and answered questions about the CA presented in one situation. Unlike Study 1, we fixed the gender of the customer, and the participants were presented with only one situation randomly selected among the nine different situations (3 male, 3 neutral, and 3 female situations). The instructions specified the customer’s gender (e.g., ‘this customer is a woman’ or ‘this customer is a man’) in each situation. We used the same questionnaire about the CA’s appropriate features as in Study 1. An item was added to the post-experimental questionnaire to check if participants correctly recalled the gender of the customer. Thus, we conducted a second study, with a larger sample, in which we controlled the gender of the customer and used a between-subjects design to minimize any awareness about our hypotheses, by limiting the number of situations presented. Results Data Preparation H2a: Participants would consider the male gender more appropriate for the CA in stereotypically male situations and the female gender more appropriate in stereotypically female situations, regardless of the customer’s gender. The Appropriate Level of Warmth Participants may have inferred that the customer is a woman in stereotypically female situations and a man in stereotypically male situations. This inference could have influenced participants through unexpected processes. Research shows that people prefer CAs that look like them and have a similar gender (ter Stal et al., 2020; Bailenson et al., 2008). Hence, men would prefer masculine CAs and women would prefer feminine CAs. However, to decrease social desirability, we asked participants to take the perspective of an average customer (not their own perspective), and interestingly we found no effect of the participants’ gender. However, in order to meet the requirements of the task (i.e., rate the appropriate level of traits to increase the customer satisfaction), participants could have answered based on the two uncontrolled inferences that the customer is a woman (a man) in female (male) situations and based on their gut feeling that a female (male) customer would prefer a female (male) agent to match the customer gender. Since we want to ascertain that the gendered situations influence the perception of the agent and answers are not related to the customer gender, we decided to manipulate the customer gender in Study 2. STUDY 2 In this study, we aimed to replicate the results of Study 1 and to overcome its limitations. We presented only one gendered situation type (male, neutral, or female) using a between-subjects design, and we presented the customer as either a man or a woman. We formulate the following hypothesis: Data Preparation The dependent variables were the same as in the first study. We obtained the perceived appropriate gender of the CA (1 = male to 5 = female), an appropriate level of warmth index (mean evaluation of warmth traits from 1 = not at all to 5 = very much), and an appropriate level of competence index (mean evaluation of competence traits from 1 = not at all to 5 = very much). H2b: Participants would deem warmth features more appropriate for the CA in stereotypically female situations and competence features more appropriate in stereotypically male situations, regardless of the customer gender. H2b: Participants would deem warmth features more appropriate for the CA in stereotypically female situations and competence features more appropriate in stereotypically male situations, regardless of the customer gender. An analysis of covariance8 was run to test our hypothesis. We used four independent variables in a between-subjects model. The first independent variable (IV) was the stereotypically gendered situation, with three categories (male, neutral, female). To decompose omnibus effects, as in Study 1, we tested a linear effect H2c: We expected the effect of the stereotypically male and female situations to be moderated by sexist attitudes such that the more sexist (hostile or benevolent) the participant is, the more they would rely on gender stereotypes in their Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 9 a significant effect of C1, F(1, 246) = 17.44, p < .001, η²p= .06, B = 0.17, 95% CI [0.09, 0.25], but not of C2, p = .15. The appropriate gender increased linearly toward femininity when passing from stereotypically masculine situations (M = 2.84; SD = 0.54) to stereotypically female situations (M = 3.23; SD = 0.47). Moreover, we observed a significant interaction between the stereotypically gendered situation and hostile sexism, F(2, 246) = 3.27, p = .039, η²p= .02. Specifically, the interaction between hostile sexism and C1 was significant, F(1, 246) = 5.22, p = .023, η²p = .02, B = 0.09, 95% CI [0.012, 0.17], but not the interaction with C2, p = .23. As predicted, the higher the participant’s hostile sexism score of the participant, the more the gendered situation influenced their evaluation of the appropriate CA gender (Figure 4). Data Preparation The gender of the customer had no main effect, F(1, 246) = 0.76, p = .38, no significant interaction effect with the situation, F(1, 246) = .40, p = .66, nor with the ambivalent sexism (Fs < 1). with a contrast C1 (female = +1; neutral = 0; male = –1) and a residual contrast C2 (female = –1; neutral = +2; male = –1). The second IV was the customer’s gender, with two categories (male or female). Benevolent sexism (centred) and hostile sexism (centred) were the third and fourth IVs entered in the model as continuous variables. All main effects, one-way, two-way, and three-way interactions were tested (complete model). Control variables (participants’ gender, suspicion level, and attitudes towards CAs) and their interaction with the gendered situation IV were added and their impact was tested for each measure (as suggested by the comparison model approach of Judd et al., 2017). The results did not show a significant impact of the control variables on the explained variance and interactions with the IVs of interest, and they were discarded from the analysis. Similarly to the first study, all measures showed acceptable internal constancy (see Table 2). Two outliers (one for the appropriate gender and one for the appropriate level of warmth) were detected with the cook’s distance and discarded from the analysis (see Judd et al., 2017). The analysis was performed on 270 participants for the appropriate gender and appropriate warmth and 271 for the appropriate competence. MEAN (SD) CRONBACH’S ALPHA Hostile sexism 2.32 (1.14) .90 Benevolent sexism 2.84 (1.12) .83 Appropriate competence 4.47 (0.47) .69 Appropriate warmth 4.00 (0.63) .70 Attitudes toward CAs 4.46 (1.16) .88 Table 2 Means (SD) and Cronbach’s alpha of variables included in the model (study 2). Discussion of Study 2 The goal of this second study was to replicate the results of the first study and control the effect of the customer’s gender on the CA’s appropriate gender, warmth and competence. We manipulated the customer gender between-subjects (the customer was either a man or a woman). Given the work on CA preference, gender, and gender stereotypes (ter Stal et al., 2020; McDonnell & Baxter, 2019; Brahnam & De Angeli, 2012), we reasoned that the gender of the customer may influence the perception of appropriate features for the CA in such a way that manly features in the CA could be seen as more appropriate for male than female customers (and vice versa). While a majority of participants correctly recalled the customer gender (69% of participants did so), we did not find any main or interaction effect of the customer gender on the appropriate gender, appropriate level of warmth, or appropriate level of competence of the CA. Our results are not related to the customer gender or to any expected match between the customer gender and the conversation agent gendered features. The participants judged that female CAs were more appropriate in stereotypically female situations and male CAs were more appropriate in stereotypically male situations, even when the CA has itself no gender features. Hostile sexism moderated some of the effects such that the more hostile sexist the participants are, the more stereotypical their perceptions were. However, benevolent sexism did not predict nor moderate any effect. In both studies, competence traits were rated as more appropriate in stereotypically male situations than in stereotypically female situations. This effect was not significantly moderated by the level of sexism in any of the studies. In Study 2, we unexpectedly found that competence was perceived as more appropriate in stereotypically male and female situations than in neutral situations. This effect did not appear in Study 1 in which all scenarios were rated in a within-subject design. This effect may be related to the change of design from within-subjects to between-subjects designs. Also, hostile sexism had a positive main effect on the appropriate competence level. Replication is needed. We used a between-subjects design in which participants were presented with one kind of situation (either a stereotypically male, or a stereotypically female, or a gender-neutral situation). The moderation effects of hostile and benevolent sexism were tested in the same way as in the first study. The Appropriate Level of Competence A significant effect of stereotypically male and female situations was found, F(2, 247) = 7.21, p < .001, η²p = .05. The decomposition of this effect indicated an effect of C1, F(1, 247) = 9.46, p < .01, η²p = .037, B = –0.10, 95% CI [–0.17, –0.03], and of C2, F(1, 247) = 5.71, p = .017, η²p = .02, B = –0.05, 95% CI [–0.09, –0.009]. This effect was not linear (Mmale= 4.62; SDmale = 0.36, Mneutral= 4.36; SDneutral = 0.45, Mfemale = 4.46; SDfemale = 0.40). Participants perceived that competence traits were more appropriate in male gendered situations than in the female ones, but also more than in the neutral ones. We also observed a significant main effect of hostile sexism on the appropriate level of competence, F(1, 247) = 7.94, p < .01, B = –0.08, 95% CI [–0.14, –0.02]. The higher the sexism of the participants, the greater the attribution of competence to the CAs. Hostile and benevolent sexism and customer gender did not significantly interact with stereotypically male and female situations, nor together. A significant effect of stereotypically male and female situations was found, F(2, 247) = 7.21, p < .001, η²p = .05. The decomposition of this effect indicated an effect of C1, F(1, 247) = 9.46, p < .01, η²p = .037, B = –0.10, 95% CI [–0.17, –0.03], and of C2, F(1, 247) = 5.71, p = .017, η²p = .02, B = –0.05, 95% CI [–0.09, –0.009]. This effect was not linear (Mmale= 4.62; SDmale = 0.36, Mneutral= 4.36; SDneutral = 0.45, Mfemale = 4.46; SDfemale = 0.40). Participants perceived that competence traits were more appropriate GENERAL DISCUSSION The purpose of this paper was to empirically test the effect of gendered situations on the perceived appropriate features (gender, warmth, and competence) of neutral CAs in a customer service context, according to the participant’s level of hostile and benevolent sexism. Several stereotypically male and female situations were presented to participants in our two studies. Their task was to rate the appropriate characteristics for the conversational agent in each situation. Some of the results were in line with our hypotheses. The Appropriate Level of Warmth The Appropriate Level of Warmth We did not observe the main effect of stereotypically gendered situations, F(2, 246) = 1.98, p = .14, and no effect of the customer gender on the appropriate level of warmth, F(1, 246) = 0.12, p = .72. Moreover, hostile sexism and benevolent sexism did not interact with stereotypical situations or with the customer gender. The Conversation Agent Appropriate Gender The analysis indicated a significant effect of the stereotypically-gendered situation on the appropriate gender of the CA, F(2, 246) = 10.06, p < .001, η²p= .0.07. As we expected, the decomposition of this effect indicated Table 2 Means (SD) and Cronbach’s alpha of variables included in the model (study 2). Figure 4 Conversational agent’s appropriate gender according to the gendered situations (represented by C1) and hostile sexism (centred). A lower value in the appropriate gender corresponds to a rather masculine gender, and a higher value corresponds to a rather feminine gender. Figure 4 Conversational agent’s appropriate gender according to the gendered situations (represented by C1) and hostile sexism (centred). A lower value in the appropriate gender corresponds to a rather masculine gender, and a higher value corresponds to a rather feminine gender. Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 10 influence of the stereotypically gendered situations on the CA’s appropriate gender observed in the first study, and its interaction with hostile sexism. We partially replicate the effect of the gendered situations on the appropriate level of competence such that competence was perceived as more appropriate in the male situations than in the female situations. However, in Study 2, we also found that competence was perceived to be more appropriate in both gendered situations than in neutral situations. We did not expect that pattern. In addition, hostile sexism predicted the appropriate level of competence such that the more sexist the participants, the more competence they expect from the CA. However, the level of (hostile) sexism did not interact with the gendered situation to predict the appropriate level of competence. Finally, we did not replicate the interaction effect of attitudes toward CAs and gendered situations on the appropriate level of warmth. The appropriate level of warmth did not vary as a matter of the gendered situations nor the level of sexism. Discussion of Study 2 The results replicated the Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 11 what a (neutral) conversation agent is and that seems to have activated hostile sexism instead of benevolent sexism. Another explanation is that the task in our studies was perhaps less prone to social desirability. In fact, participants rated CAs’ features for ‘average internet users’ and not directly for themselves, an approach supposed to decrease social desirability (e.g., Fiske et al., 2002). Hence, they may have felt allowed to express their hostile attitude rather than their more controlled and polished benevolent attitude. The last explanation could be also linked to the (lack of) suspicion of participants regarding the link between the studies. Suspicion could have led participants to control their responses and resist the influence of independent variables (as some kind of reactance effect). Here, suspicion did not moderate our results or the effects of the IVs. It appears that participants were not controlling their answers and hence let their hostile attitude do its job. We had no specific hypothesis toward hostile or benevolent attitudes, both being the two sides of the same medal. Future research should more thoroughly test when hostile vs. benevolent sexist attitudes predict people’s perceptions of robots and conversational agents. We did not find any consistent effect of gendered situations on the appropriate level of warmth traits. In Study 1, it interacted with the attitude toward the CAs to predict the appropriate level of warmth but in Study 2, it did not influence the dependent variable at all. Also, we did not find any impact of the participants’ sexism level on the appropriate level of warmth. Warmth does not seem as relevant for CAs as competence or gender and is not influenced consistently by the situation. These results are in line with previous studies showing that gender stereotypes apply to robots (e.g., Bernotat et al., 2021). We extend these results by showing that gender stereotypes apply to conversation agents with no social features. We found that the mere kind of service required from the CA is enough to trigger stereotyping. For example, searching for banking advice triggered male stereotyping of the CA while searching for a beauty device triggered female stereotyping of the neutral CA. Here, we highlight that gender stereotypes creep into the smallest of gaps and that gender stereotypes matter for the conception of CAs. Discussion of Study 2 More precisely, we show that digital customer service situations convey gendered expectations that are usually observed in social interactions between humans, and that the specific digital situation affects the CA’s expected features (i.e., their congruent gender and traits). In agreement with the CASA theory research (e.g., Feine et al., 2019), we show that the gender rules apply in a digital customer service context with nonhuman CAs. Also, we show that hostile sexism but not benevolent sexism moderates some expectations of features in CAs. Our studies differs from previous research on several points. Participants are usually asked to choose tasks to which robots or virtual agents could be suited (e.g., Bernotat et al., 2021; Eyssel & Hegel, 2012; Forlizzi et al., 2007). Our studies focused instead on the features users in real life might want in a customer service context. Moreover, we designed stereotypically male and female situations and used a gender-undefined CA, rather than using gendered visual representations of the CAs. We believe that this situation design increases the external validity of our results because the required services (e.g., banking advice vs. bank opening hours) are all plausible for every human being, male or female. These situations may happen outside the lab for all of us. Here, gender stereotyping was not triggered by specific visual representations of the CA as used in previous work (e.g., Brahnam & De Angeli, 2012; Forlizzi et al., 2007; McDonnell & Baxter, 2019). Why did hostile but not benevolent sexism moderate our effects? The first explanation could lie with the Online Disinhibition Effect (Suler, 2004), stating that people experience diminished constraints because online anonymity decreases inhibition and increases self-disclosures (Hollenbaugh & Everett, 2013; Stuart & Scott, 2021). In the same way, Brahnam and De Angeli showed that people could be abusive toward virtual agents, mostly with ‘female’ CAs, explaining this effect as agent-induced disinhibition (Brahnam & De Angeli, 2012; Brahnam, 2006; De Angeli & Brahnam, 2008). Situations in our studies are not hostile, but online stereotypical situations diminish constraints, foster disinhibition, and can prime hostile sexism attitudes, explaining the moderation effect of hostile sexism. Although hostile and benevolent sexism are interrelated (Glick & Fiske, 2001), hostile sexism could have taken over benevolent sexism in our studies. This paper raises the inevitable question of ethics in the development of robots and applications. Discussion of Study 2 Our results indicated that the cognitive biases of human interactions also apply to interactions with CAs. Previous papers have debated whether to rely on gender stereotypes when developing robots (and thus CAs) to improve the user experience (e.g., in terms of credibility and trustworthiness), or to develop neutral gendered robots (and thus CAs) to prevent cognitive biases (e.g., Eyssel & Hegel, 2012). Forlizzi and their collaborators showed that people prefer CAs corresponding to their stereotypes (Forlizzi et al., 2007). To manage both customer satisfaction and mitigation of gender stereotypes, one option may be to provide some choices regarding CA features to the users, while avoiding stereotypical features Interestingly, Bernotat et al. (2021) suggested that benevolent sexism was more socially appropriate than hostile sexism to justify their findings on the relationship between the judgment of a robot and benevolent sexism. However, they used visual representations of robots that could have activated benevolent sexism rather than hostile sexism. In our study, we only define Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 12 stereotype activation, notably in the workplace, is higher when people transgress their expected gender role (e.g., Koch et al., 2015; Prentice & Carranza, 2002). For example, the more inconsistency there is between a role in the workplace and the expected gender features, the lower performance is expected from the (human) agent (Eagly & Karau, 2002; Heilman & Parks-Stamm, 2007; Stamarski & Son Hing, 2015). In future studies, it would be interesting to test situations with varying consistency between the CA’s role (e.g., expected competence vs. warmth) and the CA’s features (e.g., masculine vs. feminine). Explicit and implicit attitudes towards this CA can be tested in relation to varying role-features’ consistency. or features that may be associated with discrimination in humans. Another solution could be to foster human characteristics without gender as an androgynous face or voice (e.g., Nag & Yalçın, 2020) to avoid repeating gender stereotypes. Indeed, it was shown that a gendered conversational agent is the target of more sexist and harrassive talk than a gender-neutral agent (e.g., Brahnam & De Angeli, 2012; Brahnam, 2006; De Angeli & Brahnam, 2008). Gender-neutral agents are less verbally-abused than gendered ones and thus gender- neutral agents do not fuel so much into stereotypes, prejudice and discrimination. They would contribute less to any normalization, banalisation, or justification of sexism in real life (see, for instance, Fox et al., 2015). Discussion of Study 2 These studies have shown that gender-undefined CAs may still be the target of gender stereotyping depending on the users’ level of hostile sexism. Gender stereotyping is so widespread that the type of required service is enough to trigger gender expectations about the agent. Given that gender-neutral agents trigger less sexual and harassive talks than female agents (e.g., Brahnam & De Angeli, 2012) and that our work shows that the mere type of customer service triggers gendered expectations from sexist users, we believe developers should limit to the minimum any feature that may reinforce gender stereotyping. Finally, another limitation of the present studies lay in the direct questionnaire that measures sexism. In the future, it would be appropriate to measure the participants’ level of sexism using implicit or indirect measures (e.g., Oliveira Laux et al., 2015) to limit the suspicion of the participants and subsequently the phenomenon of social desirability. Although our study focused only on benevolent and hostile sexism, it would be interesting to consider other moderators to understand under which conditions gender stereotypes are applied to CAs. For example, one moderator could be the acceptance of new technology, which has been related to the use of CAs (e.g., Zarouali et al., 2018; Rese et al., 2020). Indeed, several studies showed that the acceptance of new technologies depended on their conformity with social norms (see Legris et al., 2003). The more normative the technology, the more acceptance it will get. From this perspective, we can assume that participants who adhere to gender norms would accept new technologies more when the technologies convey gender stereotypes that are consistent with their sexist attitudes. In this perspective, future work could test the effect of gender stereotypes on the use and acceptance of new technologies, such as CAs, depending on whether these new technologies have social aspects that are consistent with gender stereotypes or not. DATA AVAILABILITY Author 1 is the main author. He conceived the research, designed and ran the studies, analysed the data and wrote the paper. Author 2 is the second contributor. She helped conceive the research, designing the studies, analysing the data, and writing the paper. Author 3 is the last contributor. She helped analyse the data from the first study, designing the second study, and writing the paper. Data are available here: https://osf.io/ycqrx/. The female gender norm situations were as follows: The female gender norm situations were as follows: 7. We checked normality for each model and compared the results with and without a Box-Cox transformation. The results were very similar with and without Box-Cox transformations so we decided to present the results on untransformed DVs. However, for the competence DV, the Box-Cox transformation did remove a significant interaction between hostile sexism and gendered situations that appeared with the untransformed DV. For this DV, we present the results after Box-Cox transformation. The results reported here are the least favourable in regards of our hypotheses. A customer wishes to know the opening hours of a banking establishment. The customer requests a conversation agent on the bank’s website to obtain information about the opening hours. A customer does not find a desired beauty product while browsing on a commercial website. The customer requests a conversation agent on the website to find the beauty product. 8. For this second study, we did not use a Mixed Models Analysis, because participants were not exposed to the three conditions of the stereotypically gendered situations (female, neutral and male) but only to one condition. A hairdressing appliance purchased by a customer on a commercial website has broken down. The product is still under warranty. The customer requests a conversation agent on the website to find out how to use the guarantee attached to the hairdressing appliance. LIMITS AND FUTURE STUDIES We did not check the participants’ mental representations of the conversation agent. Other studies have signalled a visual representation to participants (e.g., Brahnam & De Angeli, 2012; McDonnell & Baxter, 2019), allowing them to control the mental representation of CAs. Future studies could address this limit with more ecological situations. For example, a proper interaction with a CA could be designed on a professionally-designed customer service website to test participants’ inferences, wishes, and satisfaction. There are a wide variety of social cues, which can be classified into several categories such as verbal, visual, auditory, or invisible (Feine et al., 2019). These characteristics affect the perception of CAs, such as social presence, trust, satisfaction, and credibility (Chung et al., 2020; Araujo, 2018; de Visser et al., 2016; Verhagen et al., 2014; Demeure et al., 2011). We can suppose that stereotype activation is stronger when social cues are salient than in situations without social cue. For example, we can imagine that a female voice-based CA is a stronger female social cue than a text-based CA with a female name. It will be pertinent for future studies to compare these types of CAs according to these social cues. To conclude, we have shown in these two studies that gender stereotypes apply to the perception of CAs in a similar way to our projection of the social roles observed in reality and as has been highlighted in social psychology. We have also shown for the first time that hostile sexism moderates gender stereotypes in the perception of CAs. Although. in 2012 it was suggested that we would have robot assistants in the future (Eyssel & Hegel, 2012), in 2022 we do not yet have personal robot assistants at home. However, gender-related stereotypes in computers seem not to be a thing of the past. Researchers and professionals should work closely together to minimize negative gender biases when developing conversation agents. Another point to note in our studies is that the participants had to indicate the appropriate characteristics of CAs. The research shows that gender Pinelli et al. International Review of Social Psychology DOI: 10.5334/irsp.669 13 The authors have no competing interests to declare. The authors have no competing interests to declare. The neutral gender norm situations were as follows: This research respects the current ethical principles of psychologists and the code of conduct in effect in France. It was exempt from requiring ethical approval, as it is a non-interventional research under the Loi Jardé n°2012- 300. A customer’s new bank card has not yet arrived at their home. The customer requests a chat agent on the bank’s website in order to get information about the sending of their new card. A customer wishes to know when a product indicated as ‘out- of-stock’ on a commercial website will be available again. The customer requests a conversation agent on the website to obtain information on the product’s restocking. APPENDIX 2. https://theconversation.com/theres-a-reason-siri-alexa-and-ai- are-imagined-as-female-sexism-96430 – https://thechatbot.net/ chatbot-male-female-gender-neutral/. The male gender norm situations were as follows: The male gender norm situations were as follows: 3. We controlled the time to complete the study and whether participants were distracted during the study with a single item (‘I was distracted during the study’ from 1 = strongly disagree to 7 = strongly agree; e.g., Meade & Craig, 2012). Participants completed the questionnaire in an average of 14 minutes. We excluded participants who took more than 30 minutes (M+2SD), as well as participants who declared that they were distracted during the study (answers above 3 on the 1–7 points scale). 3. We controlled the time to complete the study and whether participants were distracted during the study with a single item (‘I was distracted during the study’ from 1 = strongly disagree to 7 = strongly agree; e.g., Meade & Craig, 2012). Participants completed the questionnaire in an average of 14 minutes. We excluded participants who took more than 30 minutes (M+2SD), as well as participants who declared that they were distracted during the study (answers above 3 on the 1–7 points scale). A client wishes to open a bank account to save their money and make investments. The client requests a conversation agent on the bank’s website to find out more about the type of financial services available to save and invest money. A customer has technical questions about the operation of a handiwork product when browsing through the section of a commercial website. The customer requests a conversation agent on the website to ask technical questions about the handiwork product. 4. The study was performed in French, and we used the neutral term ‘un(e) client(e)’, which did not specify whether the customer was male or female. 4. The study was performed in French, and we used the neutral term ‘un(e) client(e)’, which did not specify whether the customer was male or female. 5. The situations were pre-tested on 20 participants to make sure that they matched the expected gender norms. Pre-test participants were asked: To what extent would the CA have masculine or feminine characteristics? (scale of 1 = male to 5 = female) (In french: Dans quelle mesure l’agent conversationnel aurait des caractéristiques masculines ou féminines?). Situations designed to be masculine led the participant to expect more masculine CAs (M = 2.48) than the situations designed to be feminine (M = 3.7). The situations designed to be neutral fell in between (M = 3). 5. The male gender norm situations were as follows: The situations were pre-tested on 20 participants to make sure that they matched the expected gender norms. Pre-test participants were asked: To what extent would the CA have masculine or feminine characteristics? (scale of 1 = male to 5 = female) (In french: Dans quelle mesure l’agent conversationnel aurait des caractéristiques masculines ou féminines?). Situations designed to be masculine led the participant to expect more masculine CAs (M = 2.48) than the situations designed to be feminine (M = 3.7). The situations designed to be neutral fell in between (M = 3). In order to purchase a computer product, a customer wishes to receive information regarding the payment in installments offered by a commercial website. The customer requests a conversation agent on the website to obtain information about this financial service. 6. 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International Review of Social Psychology DOI: 10.5334/irsp.669 Clémentine Bry Clémentine Bry Université Savoie Mont Blanc, Université Grenoble Alpes, LIP/PC2S, 38000 Grenoble, France de Visser, E. J., Monfort, S. S., McKendrick, R., Smith, M. A. B., McKnight, P. E., Krueger, F., & Parasuraman, R. (2016). Almost human: Anthropomorphism increases trust resilience in cognitive agents. Journal of Experimental Psychology: Applied, 22(3), 331–349. DOI: https://doi. org/10.1037/xap0000092 AUTHOR AFFILIATIONS Dardenne, B., Delacollette, N., Grégoire, C., & Lecocq, D. (2006). Structure latente et validation de la version française de l’Ambivalent Sexism Inventory: L’échelle de sexisme ambivalent. L’Année psychologique, 106(2), 235–264. 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DOI: https://doi.org/10.5334/irsp.669 Submitted: 08 December 2021 Accepted: 30 May 2023 Published: 12 July 2023 COPYRIGHT: © 2023 The Author(s). This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (CC-BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. See http://creativecommons.org/licenses/by/4.0/. International Review of Social Psychology is a peer-reviewed open access journal published by Ubiquity Press.
https://openalex.org/W2942355250	https://lim.bntu.by/jour/article/download/2709/2691	Russian	null	The requirements of the international standards on traceability of measurement results at OJSC «BSW – Management Company of Holding «BMC»	Litʹë i metallurgiâ	2,019	cc-by	1,838	70 / FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY 70 / 1, 2019 70 / FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY 70 / 1, 2019 Поступила 21.01.2019 Received 21.01.2019 УДК 669. DOI: 10.21122/1683-6065-2019-1-70-72 Е. В. БОРИСЕНКО, И. В. БУЯНОВА, ОАО «БМЗ – управляющая компания холдинга «БМК», г. Жлобин Гомельская обл., Беларусь, ул. Промышленная, 37, E-mail: gmetr@bmz.gomel.by Серьезным барьером при реализации продукции на рынке является доверие к организациям, выполняющим измере- ния при испытаниях продукции, а также калибровке (поверке) применяемых средств измерений. Доверие и признание результатов измерений на международном уровне обеспечивается за счет механизма аккредитации. Для обеспечения эффективного функционирования на международном уровне организации по аккредитации заключают многостороннее соглашение, которое предусматривает использование единых стандартов при проведении аккредитации. Одно из важ- ных требований этих стандартов – реализация и демонстрация метрологической прослеживаемости, которая явля- ется инструментом для обеспечения единства измерений. е слова. Метрологическая прослеживаемость, аккредитация, основа для сравнения, иерархия эталонов, мет логический контроль, единство измерений. Ключевые слова. Метрологическая прослеживаемость, аккредитация, основа для сравнения, иерархия эта логический контроль, единство измерений. Для цитирования. Борисенко, Е. В. Выполнение требований международных стандартов по прослеживаемости резуль- татов измерений на ОАО «БМЗ – управляющая компания холдинга «БМК» / Е. В. Борисенко, И. В. Буяно- ва // Литье и металлургия. 2019. № 1. С. 70–72. DOI: 10.21122/1683-6065-2019-1-70-72. ВЫПОЛНЕНИЕ ТРЕБОВАНИЙ МЕЖДУНАРОДНЫХ СТАНДАРТОВ ПО ПРОСЛЕЖИВАЕМОСТИ РЕЗУЛЬТАТОВ ИЗМЕРЕНИЙ НА ОАО «БМЗ – УПРАВЛЯЮЩАЯ КОМПАНИЯ ХОЛДИНГА «БМК» Е. В. БОРИСЕНКО, И. В. БУЯНОВА, ОАО «БМЗ – управляющая компания холдинга «БМК», г. Жлобин Гомельская обл., Беларусь, ул. Промышленная, 37, E-mail: gmetr@bmz.gomel.by E. V. BORISENKO, I. V. BUYANOVA, OJSC «BSW – Management Company of Holding «BMC», Zhlobin city, Gomel region, Belarus, 37, Promyshlennya str. E-mail gmetr@bmz.gomel.by E. V. BORISENKO, I. V. BUYANOVA, OJSC «BSW – Management Company of Holding «BMC», Zhlobin city, Gomel region, Belarus, 37, Promyshlennya str. E-mail gmetr@bmz.gomel.by A serious barrier to the sale of products on the market is the trust in organizations that perform measurements during product testing, as well as calibration (verification) of the used measuring instruments. Confidence and recognition of measurement results at the international level is ensured through an accreditation mechanism. To ensure effective functioning at the international level, accreditation organizations enter into a multilateral agreement that provides for the use of common standards in the conduct of accreditation. One of the important requirements of these standards is the implementation and demonstration of metrological traceability, which is a tool for ensuring the uniformity of measurements. Keywords. Metrological traceability, accreditation, basis for comparison, hierarchy of standards, metrological contro surements. Keywords. Metrological traceability, accreditation, basis for comparison, hierarchy of standards, metrological control, unity of mea- surements. For citation. Borisenko E. V., Buyanova I. V. The requirements of the international standards on traceability of measurement results at OJSC «BSW – Management Company of Holding «BMC». Foundry production and metallurgy, 2019, no. 1, pp. 70–72. For citation. Borisenko E. V., Buyanova I. V. The requirements of the international standards on traceability of measurement results at OJSC «BSW – Management Company of Holding «BMC». Foundry production and metallurgy, 2019, no. 1, pp. 70–72. DOI: 10.21122/1683-6065-2019-1-70-72. Требования, устанавливаемые к продукции и условиям ее размещения на рынке, различаются в раз- ных странах. Обычно выделяют четыре типа технических барьеров, которые встают перед продукцией, выпускаемой на зарубежные рынки. Первый – собственно требования к продукции. Второй – схемы и процедуры оценки соответствия. Мало установить требования, их необходимо определенным образом проверить. Третий – методы испытаний и измерений. Четвертый – доверие к организациям, проводящим соответствующие испытания и измерения, которое и обеспечивается за счет механизма аккредитации. Мировая аккредитация разделена на два уровня: региональный (ЕА – Европейская организация по аккредитации) и глобальный (ILAC – Международная организация по аккредитации лабораторий, IAF – Международный форум по аккредитации). Международные организации поддерживают работу в духе / 71 / 71 ËÈÒÚÅ¾½ È ÌÅÒÀËËÓÐÃÈß ËÈÒÚÅ¾½ È ÌÅÒÀËËÓÐÃÈß ËÈÒÚÅ¾½ È ÌÅÒÀËËÓÐÃÈß / 1, 2019/ соглашений для усиления мер по укреплению доверия к аккредитации, независимости и компетентно- сти органов по аккредитации и аккредитованных субъектов. соглашений для усиления мер по укреплению доверия к аккредитации, независимости и компетентно- сти органов по аккредитации и аккредитованных субъектов. E. V. BORISENKO, I. V. BUYANOVA, OJSC «BSW – Management Company of Holding «BMC», Zhlobin city, Gomel region, Belarus, 37, Promyshlennya str. E-mail gmetr@bmz.gomel.by Для того чтобы обеспечить эффективное функционирование аккредитации на международном уров- не, все организации должны использовать одни и те же стандарты при оценке органов по сертификации, лабораторий и контролирующих органов. Подписание многостороннего соглашения гарантирует равноценность деятельности организаций- участниц соглашения по аккредитации с помощью непрерывной и тщательной взаимной оценки, надзо- ра и повторной оценки. В ноябре 2017 г. состоялась паритетная оценка Национальной системы аккредитации Республики Беларусь со стороны Европейской организации по аккредитации (ЕА). В состав группы по оценке ЕА входили эксперты европейских органов по аккредитации Турции, Франции, Германии, Дании, Украины, Швейцарии. В течение недели зарубежные специалисты проводили наблюдения за проводимыми Бело- русским государственным центром аккредитации (БГЦА) оценками компетентности в калибровочных и испытательных лабораториях, органах сертификации продукции, систем менеджмента и персонала. По итогу визита европейскими экспертами рекомендовано подписание БГЦА соглашения о призна- нии EA в следующих областях: • испытания ISO/IEC 17025; • калибровка ISO/IEC 17025; • сертификация персонала ISO/IEC 17024; • сертификация продукции ISO/IEC 17065; • сертификация систем менеджмента ISO/IEC 17021-1. Такая положительная оценка является немаловажным фактором для ОАО «БМЗ – управляющая ком- пания холдинга «БМК». Наше предприятие является экспортером продукции в 117 стран мира, поэтому нам важно, чтобы результаты измерений, проводимых на БМЗ при испытаниях готовой продукции или калибровке средств измерений, признавались на международном уровне. Основная цель всех метрологических служб (и метрологическая служба БМЗ не исключение) – обе- спечение единства измерений на международном уровне. Для реализации этой цели создаются между- народные эталоны и единая Международная система единиц физических величин (SI), заключаются многосторонние соглашения, предусматривающие использование единых стандартов. Одним из требований международных и национальных стандартов (ТКП 8.014, СТБ ИСО/МЭК 17025, ILAC 10:01, ILAC Р14:01), которым необходимо соответствовать для обеспечения единства и при- знания результатов измерений на международном уровне, является реализация и демонстрация метро- логической прослеживаемости. обеспечения метрологической прослеживаемости необходимо выполнение двух условий: Для обеспечения метрологической прослеживаемости необходимо выполнение двух у Для обеспечения метрологической прослеживаемости необходимо выполнение двух условий: 1 Применение при калибровке (поверке) эталонов прошедших метрологический контроль Для обеспечения метрологической прослеживаемости необходимо выполнение двух условий: 1 Применение при калибровке (поверке) эталонов прошедших метрологический контроль 1. Применение при калибровке (поверке) эталонов, прошедших метрологический к ние при калибровке (поверке) эталонов, прошедших метрологический контроль. 2. Соблюдение принципа иерархии калибровок (поверок), т. е. применение для передачи единицы физической величины эталонов, имеющих лучшие по сравнению с калибруемым (поверяемым) сред- ством измерений точностные характеристики. В качестве основы для сравнения результатов измерений служат международные и национальные эталоны, сертифицированные стандартные образцы и эталонные методики измерений. 1 СТБ 8025-2005. Система обеспечения единства измерений Республики Беларусь. Поверочные схемы. Построение и содер- жание. ТКП 8.003-2011. Система обеспечения единства измерений Республики Беларусь. Поверка средств измерений. Правила проведения работ. 2 ТКП 8.014-2012. Система обеспечения единства измерений Республики Беларусь. Калибровка средств измерений. Прави- ла проведения работ. СТБ ИСО/МЭК 17025-2007. Общие требования к компетентности испытательных и калибровочных лабораторий. ILAC 10:01/2013. Политика ILAC в области прослеживаемости результатов измерений. ILAC Р14:01/2013. Политика ILAC по неопределенности измерений при калибровке. 72 / FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY 72 / 1, 2019 / FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY / 1, 2019 Роль метрологической службы БМЗ в цепи метрологической прослеживаемости заключается в пере- даче единиц физических величин от рабочих эталонов БМЗ к рабочим средствам измерений, используе- мым в подразделениях нашего завода. Эта передача осуществляется путем проведения калибровки и по- верки рабочих средств измерений в соответствии с областью аккредитации отдела главного метролога БМЗ. Лаборатории, выполняющие калибровку, поверку или испытания готовой продукции, должны де- монстрировать, что результаты проводимых измерений являются прослеживаемыми до единиц Между- народной системы SI. Каждая лаборатория, осуществляющая поверку, калибровку или испытания, должна документально подтвердить метрологическую прослеживаемость результатов своих измерений. Для поверочной лаборатории ОГМетр ОАО «БМЗ – управляющая компания холдинга «БМК» демон- страция метрологической прослеживаемости до единиц Международной системы SI основывается на локальных поверочных схемах и документальных подтверждениях метрологической прослеживаемо- сти, указанных в свидетельствах о поверке. Документами, регламентирующими построение поверочных схем и оформление свидетельств о поверке, являются государственные стандарты и технические кодек- сы установившейся практики1. Калибровочной лабораторией ОГМетр предприятия установлена метрологическая прослеживае- мость результатов измерений посредством соотнесения их с соответствующей основой для сравнения (исходными эталонами), а также документированной непрерывной цепью калибровок, каждая из кото- рых вносит отдельный вклад в неопределенность измерений. Передача размера единиц физических ве- личин представлена в виде схем метрологической прослеживаемости по видам измерений, проводимых в аккредитованной лаборатории ОГМетр ОАО «БМЗ – управляющая компания холдинга «БМК»2. Персоналом ОГМетр разработан каталог схем метрологической прослеживаемости при передаче размера следующих единиц физических величин: длины (м); массы (кг); силы (Н); твердости (по шка- лам Роквелла, Виккерса, Бринелля); температуры (°С); тока (А). Документальным подтверждением метрологической прослеживаемости при проведении калибровки средств измерений служат предоставляемые ОГМетр БМЗ свидетельства о калибровке, соответствую- щие требованиям ТКП 8.014. Подтверждением метрологической прослеживаемости, установленной в свидетельствах о калибровке, являются декларация о том, что результаты измерений прослеживаются до единиц SI; ссылка на национальные (исходные) эталоны или эталоны, стоящие на более высоком уровне в иерархии калибровок, по которым проводилась калибровка; методика калибровки; условия проведения калибровки; неопределенность результатов измерений. Документальным подтверждением метрологической прослеживаемости при проведении поверки средств измерений служат свидетельства о поверке, соответствующие требованиям ТКП 8.003 и содер- жащие декларацию о том, что результаты измерений прослеживаются до единиц SI; ссылку на нацио- нальные (исходные) эталоны или эталоны, стоящие на более высоком иерархическом уровне, по кото- рым проводилась поверка; действующую утвержденную методику поверки; установленные метрологи- ческие характеристики средства измерений; вывод о соответствии требованиям методики поверки. E. V. BORISENKO, I. V. BUYANOVA, OJSC «BSW – Management Company of Holding «BMC», Zhlobin city, Gomel region, Belarus, 37, Promyshlennya str. E-mail gmetr@bmz.gomel.by В качестве основы для сравнения результатов измерений служат международные и национальные эталоны, сертифицированные стандартные образцы и эталонные методики измерений. Метрологическая основа для сравнения может иметь локальный, национальный, региональный или международный статус. В идеале основы для сравнения представлены значениями величин националь- ных и международных эталонов, выраженными в единицах SI. Метрологическая основа для сравнения может иметь локальный, национальный, региональный или международный статус. В идеале основы для сравнения представлены значениями величин националь- ных и международных эталонов, выраженными в единицах SI. Требования к иерархии эталонов, начиная от международных эталонов и заканчивая рабочими эта- лонами, приведены ниже. Требования к иерархии эталонов, начиная от международных эталонов и заканчивая рабочими эта- лонами, приведены ниже. Международные эталоны Воспроизводят единицу величины и служат основой для определения значений общепринятой и стабильной метрологической основы для сравнения Национальные измерительные эталоны Периодически сличаются с международными эталонами более высокой точности. Служат основой для определения значений других эталонов соответствующих величин. Не предназначены для повседневной эксплуатации. Используются для калибровки вторичных эталонов государства Вторичные эталоны Используются для калибровки рабочих эталонов Рабочие эталоны Применяются национальными законодательными метрологическими службами или калибро- вочными лабораториями для поверки либо калибровки средств измерений, используемых в тор- говле и промышленности 72 / 72 / 72 / FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY FOUNDRY PRODUCTION AND METALLURGY 72 / 1, 2019 Настоящий этап практического применения метрологической прослеживаемости на ОАО «БМЗ – управляющая компания холдинга «БМК» характеризуется интеграцией требований, установленных в меж дународных, региональных и национальных документах и оформлением схем передачи размеров единиц физических величин для демонстрации и подтверждения прослеживаемости результатов измерений.
https://openalex.org/W4389575158	https://journal.formosapublisher.org/index.php/fjas/article/download/6869/6803	Indonesian	null	Marriage of Pregnant Women Out of Wedlock According to the Shafi'i School and Compilation of Islamic Law	Formosa Journal of Applied Sciences	2,023	cc-by	7,053	Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 Marriage of Pregnant Women Out of Wedlock According to the Shafi'i School and Compilation of Islamic Law Jumrah Jamil1, Risman Murut2, Muhammad Rizal Soulisa3, Jan Labondu4, Witri M. Kasuba5, Reny Raman6, Anwar Talib7 Program Studi Ahwal Al-Syakhsiyah Jurusan Syariah STAI Alkhairaat Labuha Corresponding Author: Jumrah Jamil jumrahjamil20@gmail.com A B S T R A C T Keywords: Marriage, Pregnant Women, Out of Wedlock, Syafi'I, Compilation, Islamic Law Keywords: Marriage, Pregnant Women, Out of Wedlock, Syafi'I, Compilation, Islamic Law The aim of this research is to develop a comparison. Regarding the KHI legal framework for marrying pregnant women out of wedlock, Law Number One of 1974 and Imam Syafi'i apply. This summarizes qualitative techniques with conversation-based data collection; Descriptive data processing is used in this research process. Research findings indicate a relationship. Laws between the legal framework for marriage, pregnant women at KHI, and Imam Syafi'i. Meanwhile, individuals who mate with pregnant women make a difference. KHI Law Number 1 of 1974, it is a man who impregnates a woman. marry him if the woman becomes pregnant out of wedlock. Meanwhile, according to Imam Syafi'i, marriage is the result of pregnancy out of wedlock. Legally, a woman who is pregnant may immediately marry. regardless of whether she married the man who conceived her or not. Received : 16, September Revised : 18, October Accepted: 20, November ©2023 Jamil, Murut, Soulisa, Labondu, M. Kusuba, Raman, Talib: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. Received : 16, September Revised : 18, October Accepted: 20, November 2823 ( DOI prefik: https://doi.org/10.55927/fjas.v2i11.6869 ISSN-E: 2962-6447 https://journal.formosapublisher.org/index.php/fjas ( DOI prefik: https://doi.org/10.55927/fjas.v2i11.6869 ISSN-E: 2962-6447 https://journal.formosapublisher.org/index.php/fjas ( DOI prefik: https://doi.org/10.55927/fjas.v2i11.6869 ISSN-E: 2962-6447 https://journal.formosapublisher.org/index.php/fjas 2823 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib Pernikahan Wanita Hamil Diluar Nikah Menurut Mazhab Syafi’i dan Kompilasi Hukum Islam Jumrah Jamil1, Risman Murut2, Muhammad Rizal Soulisa3, Jan Labondu4, Witr M. Kasuba5, Reny Raman6, Anwar Talib7 Jumrah Jamil1*, Risman Murut2, Muhammad Rizal Soulisa3, Jan Labondu4, Witri M. Kasuba5, Reny Raman6, Anwar Talib7 Program Studi Ahwal Al-Syakhsiyah Jurusan Syariah STAI Alkhairaat Labuha Corresponding Author: Jumrah Jamil jumrahjamil20@gmail.com A R T I C L E I N F O A B S T R A K , y , Program Studi Ahwal Al-Syakhsiyah Jurusan Syariah STAI Alkhairaat Labuha Corresponding Author: Jumrah Jamil jumrahjamil20@gmail.com A B S T R A K Tujuan dari penelitian ini adalah untuk mengembangkan perbandingan. Mengenai kerangka hukum KHI untuk mengawini perempuan hamil di luar nikah, berlaku Undang-Undang Nomor Satu Tahun 1974 dan Imam Syafi'i. Hal ini merangkum teknik kualitatif dengan pengumpulan data berbasis percakapan; pengolahan data deskriptif digunakan dalam proses penelitian ini. Temuan penelitian menunjukkan adanya hubungan. Hukum diantara kerangka hukum menikahkan, Ibu Mengandung di KHI, dan Imam Syafi'i. Sementara itu, individu yang mengawinkan ibu hamil membuat perbedaan. UU KHI Nomor 1 Tahun 1974, Yang menghamili seorang perempuan adalah laki-laki. mengawininya jika perempuan itu hamil di luar nikah. Sedangkan menurut Imam Syafi'i berpendapat bahwa perkawinan akibat hamil diluar nikah adalah Secara hukum, seorang wanita yang sedang hamil boleh langsung menikah. terlepas dari apakah dia menikah dengan pria yang mengandungnya atau tidak. Kata Kunci: Pernikahan, wanita hamil, diluar nikah, Syafi’I, Kompilasi, Hukum Islam Received : 16, September Revised : 18, October Accepted: 20, November ©2023 Jamil, Murut, Soulisa, Labondu, M. Kusuba, Raman, Talib: This is an open-access article distributed under the terms of the Creative Commons Atribusi 4.0 Internasional. Received : 16, September Revised : 18, October Accepted: 20, November 2824 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 PENDAHULUAN Mengingat Indonesia termasuk negara hukum dengan mayoritas penduduk beragama Islam—sebuah realitas sosial—penting bagi undang- undang nasional untuk dirumuskan dengan mengacu pada hukum Islam. Oleh karena itu, ulama dan ilmuwan yang peduli terhadap Islam mempunyai peran yang sangat penting (Tono, Muttaqien, 1999:1). (Tono, Sidik & Muttaqien, Dadan (Eds.), 1999). Perkawinan adalah suatu kontrak atau perjanjian hukum antara dua pria yang sama kuatnya dan keduanya telah memenuhi standar hukum tentang keinginan dan kesiapan mereka untuk hidup bersama (Ali Murtadho, 2009: 29). Dalam masyarakat saat ini, menjalin hubungan romantis antara dua orang—yang sekarang disebut pacaran— bukanlah hal yang tidak disukai; di sekolah dasar, beberapa siswa mempunyai pacar. Saat ini, berkencan sering kali mengakibatkan hubungan pribadi pranikah atau seks non-seksual. Institusi perkawinan dan hubungan dipengaruhi oleh hubungan semacam ini. sangat menyimpang dari pedoman agama. Islam dengan jelas menyatakan bahwa pernikahan adalah ide yang baik karena memberikan manfaat bagi individu, pasangan, dan masyarakat secara keseluruhan. Salah satu nikmat dan wasiat keagungan Tuhan yang dianugerahkan kepada umat manusia adalah pernikahan. Mereka telah berjuang untuk mempertahankan kehidupan secara turun-temurun dan menjaga agama Allah di bumi pertiwi ini, yang dibuktikan dengan pernikahan mereka. (Mahalli, Ahmad Mudjab, 2002: 43). ( j ) Tujuan pernikahan adalah untuk memenuhi kebutuhan biologis, menenangkan jiwa, melindungi keturunan, dan menjunjung tinggi tradisi memikul tanggung jawab. Menciptakan keluarga yang mewujudkan rahmah (dan kasih sayang), mawaddah (cinta), dan sakinah (tenang)adalah tujuan utama sebuah pernikahan. Jika tujuan lain berhasil diselesaikan, hal ini dapat dicapai dengan sempurna. Dengan kata lain, tujuan perkawinan berfungsi sebagai metode tambahan untuk mencapai tujuan utama. Dari segi tujuan perkawinan, perkawinan yang didasarkan pada prinsip-prinsip Islam berupaya untuk menghasilkan anak-anak yang halal, bermoral serta keharmonisan dan kepuasan dalam hubungan antarmanusia. Kebahagiaan ada dalam bidang psikologis, sosial, dan keagamaan selain bidang fisik dan biologis. g Apakah ini tujuan setiap pernikahan? Idealnya, inilah situasinya. Namun sejauh yang kami ketahui, kawin paksa memang kadang terjadi. Pertama-tama, karena laki-laki harus bertanggung jawab atas perbuatannya, hal ini menyiratkan bahwa telah melakukan hal terlarang dengan seorang perempuan, baik perempuan itu sudah bertunangan dengannya atau tidak, yang bertentangan dengan hukum dan ajaran Islam. Konstitusi. Kedua, rasa malu keluarga perempuan disembunyikan melalui pernikahan. Misalnya, setelah berhubungan seks dengan seorang wanita, seorang pria mungkin tidak bertanggung jawab atas tindakannya dan mencari pria lain untuk dinikahinya. Kemudian, karena adanya ketidakseimbangan tertentu, menjadi tidak jelas apakah laki-laki yang menikahi perempuan tersebut bersedia melakukan hal tersebut. PENDAHULUAN 2825 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib Menurut hukum Islam, melakukan hubungan seks bebas adalah tindakan memalukan yang dikutuk oleh Allah karena dapat mengakibatkan perzinahan dan akibat negatif lainnya bagi pelakunya, seperti penyakit menular atau kehamilan yang tidak diinginkan. Sesungguhnya Allah telah menegaskan apa yang difirmankan- Ayat 32 surat Al-Isra memuatnya., khususnya: Terjemahnya: Lebih jauh lagi, hindarilah mendekati perzinahan karena itu termasuk hal buruk dan merupakan gagasan yang buruk. Al-Isra' : 32 dalam QS. TINJAUAN PUSTAKA Peneliti memeriksa data dari beberapa penelitian sebelumnya sebagai bahan perbandingan ketika menyusun artikel jurnal ini, dengan mempertimbangkan kelebihan dan kekurangan saat ini. Selain itu, untuk menemukan informasi teori-teori yang telah diterbitkan sebelum itu dihubungkan Peneliti juga menggunakan judul tersebut untuk mendapatkan landasan teori dalam ilmu pengetahuan. mencari informasi dari termasuk buku, tesis, jurnal, dan sumber informasi lainnya. , j , y 1. Studi yang dilakukan oleh Akad Nikah Bagi Ibu Hamil dan Anaknya adalah Memed Humaedillah. Sebuah buku ringkas yang memuat sejumlah topik terkait perkawinan dengan bahasa yang lugas dan komprehensif, termasuk bab tentang hukum akad nikah dan status anak dalam sudut pandang Islam, serta makna, tujuan, dan hubungan hukum yang mendasari akad. . y g 2. Penelitian yang dilakukan oleh “Fikih Ibu Hamil” adalah judul Yahya Abdurrahman al-Khatib. Buku ini membahas topik-topik yang berkaitan dengan kehamilan, mulai dari kesulitan-kesulitan yang berkaitan dengan puasa, shalat, dan haji. Kedua, hukum sewa rahim, perceraian, iddah, perkawinan ibu hamil, dan nafkah. Penjara dan qisas merupakan hukuman ketiga yang dijatuhkan kepada ibu hamil. g y g j p 3. Penelitian yang dilakukan “Hukum Menikahi Pezina dari Kajian Fiqih Islam” ditulis oleh Hamad Fakhri Azzam dan Khalid Ali Bani Ahmad. Berikan penjelasan apa itu zina dan nikah zina yang pertama. Langkah selanjutnya adalah menganalisis dengan membandingkan pendapat masing-masing mazhab dan membentuk pendapat yang jelas setelah mengungkapkan gambaran nikah zina beserta pendapat mazhab dan dalil-dalil yang digunakan.. y g g 4. Penelitian yang dilakukan oleh berjudul “Fikih Islam wa Adilatuhu” karya Wahbah az-Zuhaili. Hukum nikah, talak, khulu', meng-illa' istri, li'a>n, dzihar, dan masa 'iddah dijelaskan pada kitab jilid kesembilan. Penelitian ini relevan karena mencakup perempuan yang dilarang sementara. Terjemahnya: Terjemahnya: Lebih jauh lagi, hindarilah mendekati perzinahan karena itu termasuk hal buruk dan merupakan gagasan yang buruk. Al-Isra' : 32 dalam QS. j y Lebih jauh lagi, hindarilah mendekati perzinahan karena itu termasuk hal buru dan merupakan gagasan yang buruk. Al-Isra' : 32 dalam QS. j y Lebih jauh lagi, hindarilah mendekati perzinahan karena itu termasuk hal buruk dan merupakan gagasan yang buruk. Al-Isra' : 32 dalam QS. Menurut hukum Islam, melakukan hubungan seks di luar nikah diancam dengan zina. Jika seorang wanita melakukan zina saat hamil, maka Imam Madzhab Fiqih Islam akan memutuskan boleh atau tidaknya dia menikah dengan pria. Menurut mazhab yang dianut oleh para imamnya, seorang wanita hamil boleh menikahi pria yang menghamili atau bukan. Namun ada juga pendapat para Imam Madhabi yang berpendapat bahwa wanita hamil tidak boleh melakukan hubungan intim. Legalitas menikahi wanita selingkuh sedang hangat diperdebatkan di kalangan akademisi. Ulama hukum Islam Imam Al- Qurthubi menjelaskan tentang perkawinan seseorang dengan pezina sebagai berikut: “Sahabat-sahabat Nabi Ibnu Abbas mengartikan bahwa laki-laki itu mengawini perempuan pernah berzina dengannya berhak atas perkawinan yang sah. menikah, dia telah melakukan perzinahan, tetapi setelah akad nikah dilaksanakan, perkawinan itu menjadi sah.” Keyakinan serupa ditekuni oleh Abu Hanifah dan Imam Syafi'i. demikian. M.Quraish (2001: 166). Konsep perkawinan atau perkawinan diartikan dalam “Khususnya kontrak yang sangat kuat atau mitsaqan ghalidzan untuk melaksanakan dan menaati perintah Allah yang” tercantum dalam Kompilasi Pasal 2 Hukum Islam (KHI). ibadah” (Kementerian Agama Republik Indonesia, 2000). Pernikahan yang sah diwajibkan oleh agama. Karena diharapkan dapat terbentuk keluarga sakinah, mawaddah, dan warahmah melalui perkawinan yang sah. Tentu saja Allah mempunyai alasan untuk mewajibkan pernikahan. Yusuf Qadwhawi dalam hal ini mengatakan, jika perkawinan tidak diwajibkan oleh undang-undang, Tentu saja, percampuran seksual tidak pantas untuk mempertahankan eksistensi manusia dan tidak dapat dikendalikan. Jika hubungan seksual tidak dibatasi pada pria dan wanita tertentu yang sudah menikah, dan perzinahan tidak dilarang, maka keluarga yang memupuk sentimen sosial yang luhur seperti cinta dan kasih sayang tidak akan terbentuk. Tentu saja, ketiadaan keluarga akan menghambat terbentuknya masyarakat dan bahkan menghambat segala upaya untuk menciptakan sesuatu yang lebih baik dan ideal (Qardhawi, Yusuf, 2003). Dengan judul artikel tersebut yang telah dipaparkan, penulis mencoba menyampaikan pandangan permasalahan tersebut dalam kaitannya dengan perkawinan ibu hamil di luar nikah dari pernikahan. 2826 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 METODOLOGI Penelitian kepustakaan ini menggunakan metodologi kualitatif dengan menganalisis dua sumber primer yaitu KHI dan Mengandung di luar Nikah : Pendapat Mazhab Syafi'i. Penulis menggunakan teknik penelitian perpustakaan untuk menulis artikel ini. Mengumpulkan data dari beragam sumber pengetahuan merupakan gerakan di balik penulisan kajian dan tinjauan pustaka (2014, Harahap). Intinya, metode Belajar adalah pendekatan ilmu ilmiah untuk mengumpulkan data untuk kegunaan dan tujuan tertentu. Diperlukan suatu metode yang relevan dengan hasil yang diinginkan untuk mencapai tujuan tersebut. Pendekatan penulisan jurnal ini memadukan metode kualitatif dengan metodologi tinjauan pustaka. Penelitian yang menganalisis dan mengumpulkan data perpustakaan yang terdapat dalam buku atau jurnal dalam bentuk bahan penelitian dikenal dengan penelitian tinjauan pustaka (Hatch, 2002). 2827 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib HASIL PENELITIAN DAN PEMBAHASAN 11 2023: 2823-2840 dalam Undang-Undang Pertama Tahun 1974. Berdasarkan Undang-Undang Nomor 1 Tahun 1974, anak masuk dalam salah satu dari dua kategori: Anak yang berstatus hukum didahulukan. Kecil haram menempati urutan kedua. Undang- undang Nomor 1 Tahun 1974 Pasal 42 menyebutkan bahwa anak yang dilahirkan atau dikawinkan secara sah dianggap sah. UU Pertama Tahun 1974, RI, hal. 18. gg p Anak-anak dilahirkan dalam suatu pernikahan yang sah dianggap sah; namun, definisi ini tidak berlaku sepenuhnya jelas ketika mengkaji analisis anak sah yang disajikan dalam artikel ini. Mengatakan bahwa “anak yang lahir sah secara hukum dan agama” tidaklah problematis; Akan tetapi, pendefinisian “anak luar nikah” akan menimbulkan pembedaan, karena Anak hasil perzinahan adalah sebutan lain terhadap anak yag lahir dalam hubungan terlarang. Sejalan dengan Undang-undang Nomor 1 Tahun 1974 ayat 1 Pasal 43 yang menyebutkan anak muda itu siapa lahir di luar persatuan yang adil dapat menjalin koneksi data dengan ibu dan nak yang lahir sebelum pernikahan, namun anak dalam buaian dan lahir di luar perkawinan yang sah. dianggap sebagai tidak sah keluarganya, pernikahannya. Anak haram adalah seorang anak yang dibesarkan oleh seorang ibu tunggal yang mengandung di luar pernikahan. Pernikahan tidak sah diartikan sebagai hubungan antara seorang wanita dan seorang pria cakap untuk dilahirkan anak tetapi tidak menikah secara hukum sesuai dengan hukum yang berlaku dan agama masing-masing (Abd. Aziz Dahlan, 1999: 15). Seorang anak, sejalan dengan UUg Nomor 1 Tahun 1974, dianggap sah apabila salah satu ketentuantersebut terpenuhi. tiga syarat berikut: p p g y 1. Keturunan lahir dalam kesatuan yang sah, dalam salah satu dari dua skenario. Pertama, Berdasarkan pernikahan yang tidak terlarang, wanita mengahadirkan seorang anak. Kedua, artikel ini mengarah pada kesimpulan bahwa istri hamil sebelum akad nikah dan melahirkan setelah pernikahan. 2. Keturunan itu berasal kesatuan tifak menentang hukum dan agama. Misalnya istri hamil di tinggal mati. Istri mengandung anak yang sah karena perkawinannya sah. Satu-satunya ayat dalam Undang-Undang Nomor 1 Tahun 1974 Pasal 44 ayat (1), yang hampir menyampaikan makna tersebut adalah “Jika seorang suami dapat membuktikan bahwa istrinya melakukan perzinahan dan bahwa anak tersebut adalah hasil perzinahan itu, maka dia dapat menggugat keabsahan anak yang dilahirkan oleh isterinya tersebut.” dilahirkan.” karena perlakuan terlarang itu” (UU No. 1 Tahun 1974: 19). HASIL PENELITIAN DAN PEMBAHASAN A. Hukum Pernikahan Wanita Hamil Menurut KHI Terdapat penyimpangan terhadap posisi membahayakan hukum adat yang berkaitan dengan diterimanya perkawinan hamil dalam Kompilasi Hukum Islam. Sudut pandang realitas kompromi muncul ketika ikhtilaf dalam doktrin hukum dikaitkan dengan unsur psikologis dan sosiologis. Dengan menggunakan istishlah sebagai dasar, kesimpulan diambil dari berbagai faktor yang disajikan. Oleh karena itu, rumusan KHI berpendapat bahwa membolehkan perkawinan hamil lebih banyak manfaatnya dibandingkan dengan mengharamkannya berdasarkan kombinasi faktor ikhtilaf dan urf. Harahap, Yahya (2007), 42. Tujuan utama dari kebijakan perkawinan hamil KHI adalah mendapatkan perlindungan hukum yang jelas untuk anak-anak itu belum lahir ditempat negara tersebut; tujuan ini sengaja diungkapkan dengan cara yang jelas dan umum. Memberikan kebebasan kepada universitas untuk berinovasi dan mengungkap ide-ide baru melalui konstruksi logistik yang lebih kontemporer adalah tujuannya. Diharapkan: Orang tua melaporkan setiap aspek pengasuhan dan pendidikan anaknya hingga dewasa, berdasarkan prinsip maslahah murlahah dan memperhatikan seluruh aspek hukum, sosial, dan kemasyarakatan. Jika Pelaku zina diberi kesempatan untuk mengakui kesalahannya dan melakukan perbaikan dengan membentuk keluarga terhormat, aman, dan sah. negara. Memiliki keuntungan besar, terutama bagi janin yang dikandungnya, yang ayahnya terkait dengan kelangsungan hidup, kehormatan, dan masa depannya. Karena ayah dan ibu yang melakukan perbuatan tersebut adalah satu-satunya yang berdosa menurut hukum Islam, maka anak yang dikandungnya dibebaskan dari dosa. Namun membolehkan perempuan hamil untuk menikah mempunyai keuntungan sekaligus berpotensi mendorong perbuatan-perbuatan yang diharamkan agama (mafsadatan), seperti keinginan berselingkuh sebelum menikah. Menjamurnya multimedia yang telah menjangkau desa-desa paling terpencil sekalipun merupakan faktor utama terjadinya tindakan-tindakan yang menyimpang dari norma-norma sosial. Remaja yang memiliki akses mudah terhadap pornografi aksi dan pornografi diketahui mudah mengendalikan emosi dan hasratnya sehingga berujung pada pergaulan bebas yang berdampak pada kehamilan di luar nikah. B. Pernikahan Wanita Hamil Menurut UU No. 1 Tahun 1974 B. Pernikahan Wanita Hamil Menurut UU No. 1 Tahun 1974 UU Nomor 1 Tahun 1974 menyatakan seluruh undang-undang terkait perkawinan sebelumnya inkonstitusional. Menurut Soemiyati (1986: 2), 2. Penerapan hukum perdata (Burgerlijk wetbook), hukum perkawinan, dan hukum perkawinan bagi umat Kristiani Indonesia, di Indonesia dan undang- undang ini menjelaskan hal tersebut dengan mengatur aturan tambahan yang mengatur keseimbangan. Penjelasan mengenai itu diberikan Pasal 66 UU Perkawinan. dianggap tidak akurat. Undang-Undang Nomor 1 Tahun 1974 membolehkan perkawinan hamil, meski tidak dijelaskan secara rinci. Kesimpulan ini dibuat kemudian. mempertimbangkan Undang-Undang Nomor 1 Tahun 1974. Hanya status anak hasil perkawinan di luar nikah yang tercakup 2828 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. HASIL PENELITIAN DAN PEMBAHASAN Yang dimaksud dengan anak zina yaitu yang belum lahir atau hasil Perselingkuhannya menyebabkan dia hamil., atau bayi yang lahir karena perzinahan, di luar nikah “anak hasil zina” dalam pembahasan ini, sesuai dengan pengertian dan pendekatan terhadap arti istilah yang diberikan di atas. 2829 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib C. Hukum Wanita Hamil Dinikahkan Menurut Imam Syafi’i y Tidak disebutkan secara tegas dan jelas dalam kitab fiqih Syafi'i Al-Umm bahwa menikah dengan wanita hamil dilarang. Kesimpulan tersebut diambil setelah mempertimbangkan posisi fiqih Syafi'i terhadap pernikahan hamil. Nikah hamil menurut fiqih Syafi'i adalah bersatunya seorang perempuan hamil dengan laki-laki menjadi pasangannya yang tepat, padahal perempuan tersebut belum dalam status perkawinan atau masa iddah. Jilid 8, halaman 358. Menurut Sayyid Sabiq, sependapat Imam Hanafi dengan hukum yang membolehkan pernikahan dengan wanita hamil, dengan mengatakan diperbolehkan menikahi wanita yang berzina tanpa menunggu lewatnya masa iddah. Lebih jauh lagi, Syafi'i membolehkan pernikahan dengan wanita yang berzina meskipun mereka sedang hamil, karena kehamilan tersebut tidak menjadikan pernikahan itu haram. Sayyid Sabiq (2000) hal. 150 j p yy q ( ) Selanjutnya Imam Syafi'i menambah pandangan Hasbullah Bakry tentang hukum mengawini wanita hamil dengan menyebutkan bahwa wanita hamil yang tidak menikah akan dikenakan sanksi jika kehamilannya bukan kehamilan iddah. Iddah adalah sebutan bagi seorang janda yang suaminya meninggal dunia pada saat ia hamil atau yang suaminya menceraikannya namun ternyata ia hamil (Hasbullah BAkry, 1990:201-202). ( y ) Surat An-Nur (24): 3 memberikan landasan hukum yang didirikan Imam Syafi'i untuk membolehkan perkawinan bagi calon ibu. Dikatakan ini: Terjemahnya: Terjemahnya: j y Diharamkan bagi mukmin bagi pezina laki-laki untuk menikah dengan orang lain selain pezina menikah dengan pezinah, pemusyrik menikah dengan pemusyrik. An-Nur (QS.24): 3. Imam Al-Qurthubi yang berpendapat bahwa sahnya perkawinan seseorang dengan wanita yang pernah berzina dengannya, sependapat dengan pandangan Imam Syafi'i mengenai perkawinan dalam keadaan hamil. Ya, dia pernah berzinah sebelum menikah, namun setelah akad nikah terpenuhi, perkawinan itu menjadi sah. Shihab, M.Quraish (2001: 166). Oleh karena itu jelas bahwa iddah tidak diperlukan dalam kasus kehamilan yang disebabkan oleh perzinahan, dan diperbolehkan menikahi seorang wanita yang kehamilannya disebabkan oleh perselingkuhannya dengan pria yang menyebabkan dia hamil. 2830 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 D. Hak Wali dan Waris Anak yang Lahir dari Pernikahan di Luar Nikah D. Hak Wali dan Waris Anak yang Lahir dari Pernikahan di Luar Nikah y g Bayi yang lahir baikadalah lahir dalam atau sebagai akibat dari suatu kesatuan yang sah, seperti dimaksud dalam UU Perkawinan dan Kompilasi Hukum Islam. Definisi ini berlaku juga bagi keturunan yang dihasilkan dari suatu pernikahan seorang perempuan hamil yang kehamilannya berakhir kurang dari enam bulan setelah perkawinan resminya. Undang-Undang Nomor 1 Tahun 1974 mengatur urusan anak pada Bab IX, khusus membahas status anak pada Pasal No 43, 44, dan 42. g y Pasal 102 1) Pria beristri yang ingin menolak kelahiran anak oleh istrinya, mengajukan gugatan ke Pengadilan Agama 180 hari setelah anak itu lahir, 360 hari setelah pembubaran perkawinan, atau pada saat suami mengetahui pasanganya melahirkan anak. dan terdapat di lokasi di mana pasangan lain dapat mengajukan kasusnya. ke Pengadilan Agama Setempat. g 2) Tidak diperbolehkan menolak setelah waktu yang ditentukan telah berlalu. Pasal 103 Kompilasi Hukum Islam mengatur mengenai proses pembuktian asal-usul seorang anak. Ini menyatakan: Pasal 103 Kompilasi Hukum Islam mengatur mengenai proses pembuktian asal-usul seorang anak. Ini menyatakan: Pasal 99 Pasal 99 Pasal 99 Anak yang lahir dalam hubingan sah adalah: asa 99 Anak yang lahir dalam hubingan sah adalah: y g g 1) Keturunan itu lahir dalam atau setelah suatu kesatuan hukum. ) 2) Hasil fertilisasi ekstrauterin yang sah antara suami dan istri, yang dilahirkan oleh istri 2) Hasil fertilisasi ekstrauterin yang sah antara suami dan istri, yang dilahirkan oleh istri Pasal 101 Jika pasanagn tersebut menyangkal hal tersebut sebagimana yang telah di jelaskan, dapat meneguhkan pengingkarannya dengan li’an. Pasal 42 Bayi lahir. dalam atau di luar kesatuan hukum dianggap sebagai anak sah. Pasal 43 Bayi lahir. dalam atau di luar kesatuan hukum dianggap sebagai anak sah. Pasal 43 1) Satu-satunya hubungan penting yang dimiliki anak di luar perkawinan adalah dengan ibu dan keluarganya. 1) Satu-satunya hubungan penting yang dimiliki anak di luar perkawinan adalah dengan ibu dan keluarganya. 2) Keadaan anak sebagaimana dimaksud pada ayat (1) sebelumnya, selanjutnya diatur dengan Peraturan Pemerintah. 1) Jika seorang suami dapat menunjukkan bahwa istrinya melakukan perzinahan dan bahwa anak tersebut adalah hasil perzinahan tersebut, maka ia dapat menantang keabsahan anak yang dilahirkan oleh istrinya. p g y g y 2) Atas permintaan pihak yang berkepentingan, pengadilan memberikan penetapan mengenai keabsahan anak tersebut. g y g y 2) Atas permintaan pihak yang berkepentingan, pengadilan memberikan penetapan mengenai keabsahan anak tersebut. Pasal 55 UU Perkawinan menetapkan tata cara pembuktian darimana asal usul anak sebagai berikut: (1) Satu-satunya adalah akta kelahiran yang disahkan oleh pejabat yang ditunjuk. dokumen yang dapat membuktikan asal usul anak; (2) Apabila akta kelahiran sebagaimana dimaksud pada Dalam hal ayat (1) di pasal ini tidak ada, pihak pegadilan dapat memastikan dari mana asal usul anak tersebut dari kegiatan dilakukan penyelidikan menyeluruh dan pertimbangan bukti-bukti yang memadai. persyaratan; dan (3) Badan Pencatatan Kelahiran di wilayah hukum Pengadilan yang bersangkutan akan menerbitkan akta kelahiran bagi anak yang bersangkutan berdasarkan ketentuan Pengadilan pada ayat (2) pasal ini. p Pasal-pasal ini mengatur banyak hal. Pertama, anak-anak yang lahir baik di dalam maupun di luar suatu kesatuan yang sah diakui sebagai anak yang sah. Kemungkinan besar, tidak ada dua macam keturunan: (1) yang dua jenis kelahiran: (1) dalam perkawinan yang sah, dan (2) setelah satu perkawinan. Anak yang lajang mempunyai ikatan dengan ibu dan sanak saudaranya. Sementara itu, suami berhak mengingkari keabsahan anak yang dikandungnya dari pasangannya jika ia mampu membawa bikti nyata bahwa pasangannya berselingkuh dan bayi dari hubungan terlarang itu. Status orang tua sah seorang anak dapat disertifikasi melalui pengadilan dan akta kelahiran resmi. Artikel berikut: 99, 100, 101, 102, dan 103 KHI berisikan hal yang mengatur tentang asal usul anak. 2831 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib Pasal 100 Hanya keluarga ibu mereka dan keluarga ibunya yang merupakan kerabat garis keturunan dari anak yang belum menikah). Aturan pasal 102, 101 mengatur situasi di mana pasangan dapat menggugat keabsahan seorang anak dan langkah-langkah yang perlu diambil dalam kasus tersebut. Pasal 103 1) Hanya akta kelahiran atau dokumen resmi lainnya yang dapat mengungkapkan asal usul seorang anak. 2) Apabila tanggal kelulusan, atau dokumen lain sebagaimana dimaksud pada ayat (1) tersebut tidak tersedia, Pengadilan Agama itu dapat memastikan latar belakang anak tersebut melalui pengecekan menyeluruh itu didukung dengan bukti-bukti yang dapat dipercaya. 3) Kantor pencatatan kelahiran di lingkungan Pengadilan Agama menerbitkan akta kelahiran anak berdasarkan penetapan Pengadilan Agama pada ayat (2). 3) Kantor pencatatan kelahiran di lingkungan Pengadilan Agama menerbitkan akta kelahiran anak berdasarkan penetapan Pengadilan Agama pada ayat (2). Pasal-pasal tersebut memperjelas bahwa menurut perkawinan campur, anak yang dilahirkan di luar perkawinan hanya mempunyai hubungan kekerabatan dengan ibu dan keluarganya melalui pewarisan bersama; mereka tidak diterima oleh hukum dalam anak sah. Hal serupa juga dipertegas dalam Pasal 186 KHI yang mengatakan bahwa “Anak yang belum menikah hanya bisa menjalin hubungan” warisan timbal balik bersama ibu dan kerabatnya.” Selain itu, menurut pengertian pasal 186, keturunan yang dikandung di luar perkawinan adalah mereka yang lahir di luar perkawinan yang sah atau akibat hubungan yang tidak sah. 2832 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 Pada akhirnya Undang-undang Nomor 1 Tahun 1974 jika dicermati peraturannya, status keperdataan seorang anak hanya sebatas ibu dan keluarganya. Timbulnya Biasanya, otoritas orang tua mengacu pada tugas dan hak yang dimiliki orang tua terhadap anak-anaknya. Anak haram hanya mempunyai hubungan dengan ibu dan keluarganya, yang berakibat pada hak dan kewajiban. Inilah implementasinya. Dari pernyataan tersebut dapat ditarik kesimpulan bahwa pengertian mukhalafah adalah anak dan ayah kandungnya tidak mempunyai hubungan keperdataan berdasarkan keturunan, hak timbal balik, dan kewajiban. Meskipun asas-asas bapak biologi ini tidak disebutkan secara eksplisit dalam beberapa pasal, namun dapat tersirat bahwa Undang-Undang Nomor 1 Tahun 1974 dan Kompilasi Hukum Islam hampir sama. Menurut hukum Islam yang ada, seorang suami/pria bebas menyangkal bahwa anak yang dikandungnya bukanlah anaknya sendiri, asalkan ia mampu memberikan buktinya. Dalam mendukung penolakan diri dia harus mampu menunjukkan bahwa: 1. Istri tiba-tiba melahirkan, padahal suami belum pernah menyusuinya. 2. Sejak menikah dengan istrinya, tidak lebih dari enam anak yang dilahirkan, 2. Sejak menikah dengan istrinya, tidak lebih dari enam anak yang dilahirkan, berbeda dengan bayinya lahir seperti bayi yang telah cukup umur. 2. Sejak menikah dengan istrinya, tidak lebih dari enam anak yang dilahirkan, berbeda dengan bayinya lahir seperti bayi yang telah cukup umur. Pasal 103 Para imam mazhab berbeda pendapat mengenai status kelahiran Keturunan yang dihasilkan dari penyatuan ibu hamil: Para imam mazhab berbeda pendapat mengenai status kelahiran Keturunan yang dihasilkan dari penyatuan ibu hamil: 1. Para ulama sepakat bahwa ibulah yang diberi hak asuh atas anak yang dilahirkan setelah dewasa, bukan ayah. Sebagaimana dikemukakan Ibnu Rusyd dalam Bidayatul Mujtahid, “para ulama sepakat bahwa anak-anak hasil masa dewasa tidak dititipkan kepada bapaknya, kecuali yang terjadi pada zaman jahiliah”? p j 2. Menurut Imam Syafi'i, seorang anak mempunyai hubungan kekerabatan dengan suaminya apabila perkawinan berlangsung lebih dari enam bulan dan usia kehamilan paling awal adalah enam bulan. Sebaliknya, silsilah anak disambungkan dengan ibunya jika usianya kurang dari enam bulan. Imam Syafi’i menurut Ibnu Rusyd menyatakan: “Ketika seorang pria menikahi seorang wanita tanpa bercampur dengannya atau bercampur dengannya setelah akad, maka perempuan Itu menghasilkan seorang anak dalam enam bulan setelah pernikahan, tidak dari percampuran tersebut. kemudian, jika ibu menunggu lebih dari enam bulan untuk melahirkan, maka penghitungan kelahiran anak oleh laki-laki itu batal. mengawininya. Memperhatikan pandangan tersebut dalam konteks pengertian teknis perkawinan (nikah) yang menyatakan bahwa perkawinan adalah akad yang mengharamkan berhubungan badan. Akibatnya, jika seorang perempuan hamil sebelum perjanjian itu dipenuhi, maka kehamilannya tidak dihormati, dan laki-laki mana pun boleh menjadi ayah dari anak tersebut karena anak tersebut tidak mempunyai keturunan dari bapaknya. tapi ibunya adalah satu-satunya leluhurnya; 2833 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib 3. Menurut Imam Hanafi, hubungan anak adalah dengan suami ibu jika ibu mengandung anak dalam keadaan menikah. Oleh karena itu, setiap anak yang dilahirkan akan memiliki keterkaitan dengan laki-laki pemilik benih tersebut, menurut Imam Hanafi. Klausul ini ditunjukkan dengan pola pikir yang memandang pernikahan sama dengan hubungan seksual. Oleh karena itu, bayi tersebut merupakan keturunan dari pezina selama masih ada aktivitas seksual yang berujung pada lahirnya anak. Dengan demikian, anak yang dilahirkan dari perkawinan perempuan hamil tidak secara langsung dikaitkan dengan laki-laki yang mengawini ibu anak tersebut; Sebaliknya, hal ini dikaitkan dengan mereka yang menuai benih, yang berarti bahwa hal tersebut dapat juga dikaitkan dengan seseorang yang bukan ibu dari anak tersebut. 3. Menurut Imam Hanafi, hubungan anak adalah dengan suami ibu jika ibu mengandung anak dalam keadaan menikah. Oleh karena itu, setiap anak yang dilahirkan akan memiliki keterkaitan dengan laki-laki pemilik benih tersebut, menurut Imam Hanafi. Klausul ini ditunjukkan dengan pola pikir yang memandang pernikahan sama dengan hubungan seksual. Haknya y Ketentuan mengenai batas minimal usia lahir dalam hukum Islam mengatur bahwa anak yang dihasilkan oleh ibu yang mempunyai pasangan adalah berumur enam bulan. Menurut Muhammad Jawad Mughniyah, jika salah sahabat putus dengan pasangannya setelah sekian lama berpacaran, maka pasangan tersebut kemudian akan berselingkuh dengan orang lain setelah perkawinan berakhir. Selanjutnya, setelah setengah tahun perkawinan dengan suami orang tua yang lain, tetapi kurang dari setahun jika berkaitan dengan perkawinan anak dengan suami orang tua, tetapi tidak lebih dari setengah tahun dari jumlah minimal mengandung, maka bayi tersebut di pertanyaan diajukan kepada pria orang tua. Namun, silsilah bayi tersebut tetap terhubung dengan suami kedua jika anak tersebut lahir setelah pasangan tersebut menikah selama enam bulan. (2011: 387Mughniyah, Muhammad Jawad) Enam bulan kemudian, Imam Malik dan Syafi'i menyatakan bahwa anak tersebut telah lahir.. menikah. orangtuanya, dianggap berasal dari ayahnya. Ibu diberikan hak asuh atas anak tersebut jika lahir sebelum enam bulan. Imam Abu Hanifah tidak sependapat dengan mengatakan bahwa anak haram tetap dianggap ayahnya sebagai anak sah. (Hasan, M.Ali, 1997:81) Landasan hukum masa kehamilan minimal enam (enam) bulan setelah seorang ibu menikah terdapat dalam surat Al-Luqman (31): 14: Pasal 103 Oleh karena itu, bayi tersebut merupakan keturunan dari pezina selama masih ada aktivitas seksual yang berujung pada lahirnya anak. Dengan demikian, anak yang dilahirkan dari perkawinan perempuan hamil tidak secara langsung dikaitkan dengan laki-laki yang mengawini ibu anak tersebut; Sebaliknya, hal ini dikaitkan dengan mereka yang menuai benih, yang berarti bahwa hal tersebut dapat juga dikaitkan dengan seseorang yang bukan ibu dari anak tersebut. Terjemahnya: j y Dan Kami perintahkan kepada manusia (berbuat baik) kepada dua orang ibu- bapanya; ibunya telah mengandungnya dalam Keadaan lemah yang bertambah-tambah, dan menyapihnya dalam dua tahun. Bersyukurlah kepadaku dan kepada dua orang ibu bapakmu, hanya kepada-Kulah kembalimu. (QS. Al-Luqman (31) : 14). 2834 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 Bagi yang ingin menyelesaikan masa menyusui berlangsung untuk jangka waktu 24 bulan dua tahun.Hapus periode setengah tahun jika periode tiga puluh bulan lebih pendek dari periode dua puluh empat bulan. Itu adalah durasi minimum kehamilan. (Zainal Abidin dan Ibnu Mas'ud, 2007: 414) Para akademisi yang mengkaji status anak haram sepakat bahwa anak tersebut masih mempunyai ikatan kekeluargaan dengan ibunya. Seluruh kebutuhan keluarga, baik material maupun spiritual, dipenuhi oleh ibu dan keluarganya. Begitu pula halnya dengan hak untuk mewarisi. (Juz V : 357 Ibnu Rusyd; Bidayah al- Mujtahid) j Akibat hukum yang timbul dari status anak haram pada golongan kedua yang disamakan dengan anak hasil zina dan anak hasil zina: 1. Dia tidak memiliki hubungan kekeluargaan dengan ayahnya. Hubungan anak dengan ibunya hanya sebatas garis keturunan saja. Meski sang ayah tidak wajib menafkahi anaknya, namun anak tersebut tetaplah anak kandungnya. Akibatnya hubungan yang dihasilkan hanya bersifat manusiawi dan tidak sah. 2. Oleh sebab satu dari hal tersebut pewarisan adalah ikatan hubungan, maka tidak ada saling pewarisan dari pihak ayah. 3. Bayi dari hungan terlarang tidak boleh mempunyai ayah yang bertindak sebagai walinya. Ayah kandung tidak berhak mengawini anak haramnya apabila anak haram tersebut telah dewasa, telah bertunangan dan akan menikah, dan seorang perempuan (Amir Syarifuddin, 2002: 195). Anak yang belum menikah tidak mendapat warisan; sebaliknya, mereka berhubungan dengan ibu mereka. Disamakan dalam hadis Nabi sebagai berikut: Anak yang belum menikah tidak mendapat warisan; sebaliknya, mereka berhubungan dengan ibu mereka. Disamakan dalam hadis Nabi sebagai berikut: Dengan kata lain, menurut ‘Amr bin Syu’aib yang mengetahui hal tersebut dari ayah dan kakeknya, Sebenarnya Kata Nabi SAW bahwa “Siapa pria yang berselingkuh dengannya? budak atau Pertama, perempuan merdeka ia akan mendapat anak dari hubungan terlarang, dan ia tidak mendapat warisan.” (Hadis yang Dihimpun) Hukum Nailul Author, 2001; terjemah 2068). Pria boleh menikah dengan gadis dibawah umur, cucu perempuan, saudara perempuan, atau anak sepupunya yang merupakan hasil perzinahan, demikian karena ia tidak mempunyai hubungan garis keturunan secara syariah dengan wanita- wanita tersebut. Akibatnya terjadi pembedaan antara Imam Syafi'i dengan Kompilasi Hukum Islam, Undang-undang Nomor 1 Tahun 1974. 1. Persamaan Hukum Sesuai ayat pertama Pasal 53 Kompilasi Hukum Islam, “seorang wanita hamil yang belum menikah dengan orang lain, dapat menikah dengan lelaki yang mengawinkannya." Republik Indonesia, Nomor 1 Tahun 1974 tentang KHI dan Hukum Islam, hal. 245. Dalam Kompilasi Hukum Islam sudah jelas bahwa sahnya mengawini wanita hamil sebelum menikah. Ungkapan “seorang wanita hamil dapat dikawinkan” muncul pada Pasal 53 Ayat 1 dan memunculkan kesanggupan tersebut. Menurutnya, perempuan lajang hanya bisa menikah dengan pria yang menjadi penghamilinya; pada saat itu, anak tersebut lahir dan diakui sebagai miliknya. Dari pengertian seperti hal ini terlihat bahwa pendangan Kompilasi Hukum Islam membolehkan perkawinan dalam keadaan mengandung anak. Namun menurut pendapat Imam Syafi'i, menikahi perempuan hamil diperbolehkan oleh sebab tidak serta merta pernikahannya dilarang karena dia melakukan perzinahan. (Sabiq, Asyraf : 150) Berdasarkan temuan perbandingan tersebut, maka dapat dikatakan bahwa jika dilihat dari sisi hukum formil—yang memperbolehkan pernikahan bagi perempuan yang belum menikah—Kompilasi Hukum Islam dapat dibandingkan. Dikatakan demikian karena Imam Syafi'i dan Kompilasi Hukum Islam menganut keyakinan bahwa iddah itu ada. Dengan demikian, penulis mengambil kesimpulan bahwa pandangan pendapat Imam Syafi’i masih didukung oleh pendapat Hukum Islam itu yang memperbolehkan pernikahan di luar nikah bagi ibu hamil. Terjemahnya: Dalam Kompilasi Hukum Islam dan Undang-Undang Nomor 1 Tahun 1974 diatur bahwa tidak ada tindakan segera yang diperlukan untuk lahirnya seorang Imam Syafi'i. seorang anak dan warisan itu terbatas pada pihak ibu sepanjang perkawinan itu terjadi dan garis keturunan anak itu ada hubungannya dengan garis keturunan ibu. Sedangkan enam bulan merupakan waktu paling cepat bagi seorang anak untuk dilahirkan menurut Imam Syafi'i. Garis keturunan anak tersebut dikategorikan sebagai garis keturunan ibu dan hanya mewarisi dari ibunya apabila ia dilahirkan kurang dari enam bulan. 2835 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib 2. Perbandingan Hukum Pasal 53 Kompilasi Hukum Islam pada ayat pertamanya menyatakan bahwa “seorang perempuan asing yang sedang hamil dapat dikawinkan dengan laki- laki yang menghamilinya”. (UU Nomor 1 Tahun 1974 tentang Kompilasi Hukum Islam dan Perkawinan, hal. 245) Berdasarkan bunyi pasal tersebut, maka jika seorang perempuan hamil di luar nikah, maka laki-laki yang mengandungnya dapat mengawininya. Kompilasi Hukum Islam menegaskan bahwa mengawini perempuan hamil diperbolehkan apabila laki-laki yang menghamilinya juga yang boleh mengawininya. Dalam hal ini, nampaknya benih yang menghasilkan anak tersebut masih berasal dari laki-laki tertentu; hanya saja wanita tersebut bukanlah istrinya saat benih itu dimasukkan ke dalam rahimnya. Karena belum mengikat ikatan perkawinan, maka penafsiran Kompilasi Hukum Islam ini jelas lebih mengutamakan sudut pandang luar dibandingkan hakikat realitas yang sebenarnya (benih itu milik sendiri), bukan pada formalitas luar (tidak memandang benih). Undang-Undang Pertama Tahun 1974menyatakan bahwa persatuan mereka menghormati keyakinan agama atau filosofi pribadi setiap pasangan dianggap sah. Persyaratan hukum mengamanatkan agar setiap perkawinan didaftarkan. 2836 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 “Anak Anak yang lahir dalam atau sebagai akibat perkawinan yang sah dianggap sebagai anak sah.,” menurut Pasal 42 Undang-Undang Nomor 1 Tahun 1974 yang mengatur tentang penentuan keturunan berdasarkan perkawinan yang sah.”. Aturan-aturan Secara tegas dikatakan bahwa orang muda yang dilahirkan oleh pasangan suami istri berhak diakui sebagai anak sah. Sang istri hamil dan melahirkan setelah akad nikah resmi dilaksanakan. Sebaliknya jika seorang perempuan hamil sebelum penandatanganan akad nikah dan melahirkan setelah akad nikah, maka anak tersebut dianggap tidak sah. Menurut Imam Syafi'i, membolehkan nikah hamil baik dengan laki-laki yang mengandung atau tidak—adalah cara terbaik untuk mengetahui apakah seorang wanita hamil di luar nikahnya. Menurut Hasbullah Bakry, kemampuan tersebut dapat dilihat dari sudut pandang Imam Syafi'i yang mengatakan: Sedangkan karena belum pernah menikah, maka anak perempuan yang hamil tidak mengalami masa iddah. Seorang pria dapat mengajukan lamaran kehamilan kepada seorang wanita kapan saja. Mazhab Syafi'i melebur dengan dua tradisi filosofis tambahan, Hanafi dan Maliki. Menurut Syafi'i, baik laki-laki maupun perempuan diperbolehkan. menikah dengan orang yang sedang hamil, dan setelah menikah boleh disetubuhi meskipun bukan ibunya yang menghamilinya.” (Hasbullah Bakra (202). Kejanggalan ditemukan saat membandingkan Undang-undang Nomor 1 Tahun 1974, Kompilasi Hukum Islam, dengan Imam Syafi'i. Kompilasi Hukum Islam mengharuskan laki-laki yang menghamili seorang perempuan untuk mengawininya di luar nikah. Wanita yang berselingkuh dan hamil diperbolehkan menikah di samping pria yang mengandungnya, sesuai dengan Undang-Undang Pertama Tahun 1974. 2. Perbandingan Hukum Seorang perempuan dapat menikah secara sah apabila ia menikah dengan laki- laki yang dihamilinya melalui perzinahan; anak yang lahir dari perkawinan ini membuktikan orang tua dari anak tersebut. g Imam Syafi'i mengklaim pria mana pun boleh menikah. perempuan hamil yang belum menikah. Oleh karena itu, laki-laki boleh saja setuju untuk menyewakan perempuan tersebut dan kemudian mencampurkannya, meskipun perempuan tersebut sedang hamil. Salah satu faktor Alasan lainnya adalah perempuan yang belum menikah tidak mendapat iddah ketika hamil. Meskipun kehamilan tersebut diketahui pada saat suami janda tersebut mengajukan gugatan cerai, namun Kehamilan yang digambarkan sebagai iddah hanya terjadi pada seorang janda yang suaminya meninggal setelah kehamilannya. Sementara itu, untuk mencegah terjadinya percampuran keturunan, Undang-undang Nomor 1 Tahun 1974 dan Kompilasi Hukum Islam memberikan batasan terhadap laki-laki yang sedang hamil. mengawini perempuan hamil yang belum menikah. Terdapat perbedaan antara Imam Syafi'i dengan Kompilasi Hukum Islam, Undang-Undang Nomor 1 Tahun 1974, tentang Warisan dan Keturunan. Anak tidak menerima apa pun selain warisan dari pihak ibu. tindakan segera yang diperlukan atas kelahiran anak tersebut, menurut Kompilasi Hukum Islam dan Undang-Undang Nomor 1 Tahun 1974, dengan syarat dilangsungkan perkawinan. Sedangkan enam bulan adalah tanggal lahir paling awal menurut Imam Syafi'i. Seorang anak dianggap berasal dari garis keturunan ibu dan hanya dapat mewarisi dari ibu jika usianya kurang dari enam bulan. 2837 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib KESIMPULAN DAN REKOMENDASI Berikut kesimpulan yang dapat diambil dalam pembahasan ini berdasarkan uraian yang telah selesai. Sesuai dengan Kompilasi Hukum Islam, berikut hukum mengenai perkawinan hamil: Disebutkan dalam Kompilasi Hukum Islam bahwa seorang perempuan boleh menikah jika dia hamil meskipun dia tidak mempunyai iddah atau status perkawinan karena perkawinan sah sebelumnya dengan laki- laki yang menyebabkan terjadinya pembuahan seorang wanita. Namun Undang- Undang Nomor 1 Tahun 1974 tidak menjelaskan secara jelas boleh atau tidaknya seorang perempuan hamil dikawinkan. Informasi mengenai anak luar nikah tersedia berdasarkan UU No. 1. Imam Syafi'i menyatakan bahwa pernikahan diartikan sebagai bersatunya seorang perempuan hamil dengan seorang laki-laki, baik yang menghamilinya maupun yang tidak, padahal perempuan tersebut belum berstatus kawin atau dalam masa iddah karena adanya keabsahan pernikahannya dengan laki-laki tersebut. Undang-undang ini Artinya perempuan yang sedang hamil boleh menikah dengan laki-laki yang menghamilinya., namun Anak-anak pasangan itu tidak sah dan ibunya mewarisi garis keturunan. Berikut ini perbandingan hukum perkawinan hamil antara Imam Syafi'i, KHI dan Undang-undang Nomor 1 Tahun 1974: KHI menyatakan bahwa laki-laki yang menghamili perempuan adalah yang mendapat menikah dengannya di luar Najah. Baik Komisi Hukum Islam maupun Undang-Undang Nomor 1 Tahun 1974 dengan tegas menyatakan bahwa tumbuhnya bulu wajah pada ibu hamil merupakan tanda yang menyebabkan ibu hamil tersebut menjadi seorang wanita. Di sisi lain, Imam Syafi'i menegaskan, laki-laki yang mengandung anak dari perempuan yang sudah menikah atau laki-laki yang sedang mengandung masih cukup muda dapat dianggap perawan. Undang-undang Nomor 1 Tahun 1974 dan Kompilasi Hukum Islam menyatakan bahwa tidak ada batas atas usia kapan anak dapat dilahirkan dan ibulah yang menentukan nasib anak-anak yang belum menikah yang dilahirkan oleh ibu hamil. Sementara Imam Syafi'i menjelaskan, bayi yang baru lahir bisa lahir paling cepat enam bulan. Jika anak tersebut berumur kurang dari enam bulan, maka garis keturunannya berpindah ke garis keturunan ibu. Sedangkan Imam Syafi'i tidak mewarisi apa pun. sesuai Undang- Undang Nomor 1 Tahun 1974 tentang Kompilasi Hukum Islam yang mengatur tentang hak waris bagi anak yang lahir di luar perkawinan. Berikut ini perbandingan hukum perkawinan hamil antara Imam Syafi'i, KHI dan Undang-undang Nomor 1 Tahun 1974: KHI menyatakan bahwa laki-laki yang menghamili perempuan adalah yang mendapat menikah dengannya di luar Najah. Baik Komisi Hukum Islam maupun Undang-Undang Nomor 1 Tahun 1974 dengan tegas menyatakan bahwa tumbuhnya bulu wajah pada ibu hamil merupakan tanda yang menyebabkan ibu hamil tersebut menjadi seorang wanita. UCAPAN TERIMA KASIH Dengan bantuan berbagai pihak, penelitian ini dapat terselesaikan. Artikel ini dianggap layak untuk diterbitkan karena hal-hal sebagai berikut: dosen STAI Alkhairaat Labuha, Wakil Ketua I, II, dan III, Ketua STAI Alkhairaat Labuha, dan Berkat Formosa Journal of Applied Sciences (FJAS), hasilnya kini dapat dipublikasikan. karyanya. KESIMPULAN DAN REKOMENDASI g g y g p Adapun Saran yang diberikan pada naskah ini adalah sebagai berikut: Adapun Saran yang diberikan pada naskah ini adalah sebagai 1. Penulis berpesan kepada remaja untuk ekstra hati-hati dalam berinteraksi dengan jenis kelamin lainnya karena standar dan etika sering dilanggar di era globalisasi saat ini. 1. Penulis berpesan kepada remaja untuk ekstra hati-hati dalam berinteraksi dengan jenis kelamin lainnya karena standar dan etika sering dilanggar di era globalisasi saat ini. 2. Anggota KPU disarankan untuk mendapat kuasa hukum dari Pengadilan Agama dan Kantor Agama, mengingat mengawini perempuan Kehamilan di luar nikah diakui sebagai produk sah menurut Kompilasi Hukum Islam. dengan beragam penerapan serta akibat. masyarakat umum. 2838 Formosa Journal of Applied Sciences (FJAS) Vol. 2, No. 11 2023: 2823-2840 pp ( ) Vol. 2, No. 11 2023: 2823-2840 3. Masyarakat Islam Indonesia disarankan untuk mendidik generasi mudanya untuk mencegah pergaulan bebas remaja yang berujung pada pernikahan muda. Ketika anak seorang Ketika seorang wanita yang belum menikah hamil, dia harus menikah dengan seorang pria mengandung anaknya untuk menyembunyikan rasa malu atas kehamilannya. y 4. Perguruan tinggi, khususnya STAI Alkhairaat Labuha, dapat melakukan pendidikan hukum kemasyarakatan, khususnya mengenai Pergaulan bebas remaja dan risiko kehamilan yang tidak diinginkan. 4. Perguruan tinggi, khususnya STAI Alkhairaat Labuha, dapat melakukan pendidikan hukum kemasyarakatan, khususnya mengenai Pergaulan bebas remaja dan risiko kehamilan yang tidak diinginkan. PENELITIAN LANJUTAN J Artikel jurnal ini ditulis menggunakan metode kualitatif dalam format tinjauan pustaka. Penelitian yang menganalisis dan mengumpulkan data perpustakaan—yang terdapat dalam buku atau jurnal—dalam bentuk bahan penelitian dikenal dengan penelitian tinjauan pustaka (Hatch, 2002). Tinjauan literatur ini dapat berfungsi sebagai peta jalan untuk penyelidikan tambahan, khususnya penelitian analitis dan deskriptif untuk membantu Anda memahaminya Pernikahan Wanita Hamil Belum Menikah Mazhab Syafi'i dan Kompilasi Hukum Islam menyatakan bahwa.. DAFTAR PUSTAKA Anjelina Winda, Silvia Nova, Gitituati Nurhizrah, Program Merdeka Belajar, Gebrakan Baru Kebijakan Pendidikan, Pendidikan Dasar, Universitas Negeri Padang, Volume 5 Nomor 1 Tahun 2021, ISSN: 2614 6754 (print) ISSN: 2614-3097(online), Dela Khoirul Ainia, Merdeka Belajar dalam Pandangan Ki Hadjar Dewantara dan Relevansinya bagi Pengembangan Pendidikan Karakter, Magister Filsafat, Universitas Gadjah Mada, Yogyakarta, Indonesia, Jurnal Filsafat Indonesia, Vol 3 No 3 Tahun 2020 ISSN: E-ISSN 2620-7982, P-ISSN: 2620- 7990 Dewantara, Ki Hadjar. (2009). Menuju Manusia Merdeka. Yogyakarta: Leutika Dewantara, Ki Hadjar. (2011). Bagian Pertama Pendidikan.Yogyakarta: Majelis Luhur Pesatuan. Dwiarso, Priyo. (2010). Napak Tilas Ajaran Ki Hadjar Dewantara.Yogyakarta: Majelis Luhur Pesatuan. Haidar Musyafa. (2015). “Sang Guru”. Novel Ki Hajar Dewantara, Kehidupan, Pemikiran, Perjuangan Pendirian Taman Siswa, 1889-1959.Yogyakarta: M. Kahfi. 2839 Jamil, Murut, Soulisa, Labondu, M.Kusuba, Raman, Talib Kemendikbud. (2019). “Merdeka Belajar: Pokok-Pokok Kebijakan Merdeka Belajar”. Jakarta: Makalah Rapat Koordinasi Kepala Dinas Pendidikan Seluruh Indonesia. Lickona, T. (1991). Educating for Character. Bantam Books. Marihandono, Djoko. (2017). Rawe-Rawe Rantas Malang-Malang Putung: Jejak Soewardi Soerjaningrat Hingga Pembuangan. Jakarta: Makalah Seminar “Perjuangan Ki Hadjar Dewantara dari Politik ke Pendidikan. Merdeka Belajar Menuju Pendidikan Ideal. (2019, Desember 18). Media Indonesia. Diakses dari https://mediaindonesia.com/read/detail/278427- merdeka-belajar-menuju-pendidikan- ideal. Mudana, I Gusti Agung Made Gede, Membangun Karakter dalam Perspektif Filsafat Pendidikan Ki Hadjar Dewantara. Jurnal Filsafat Indonesia Vol. 2 No. 2 2019 h.75-81. Sekretariat GTK.(2019, November 25). Mengenal Konsep Merdeka Belajar dan Guru Penggerak. Direktorat Jenderal Guru dan Tenaga Kependidikan Kementerian Pendidikan dan Kebudayaan. Diakses dari https://gtk.kemdikbud.go.id/read-news/mengenal-konsep- merdeka- belajar-dan-guru-penggerak. Soewito, Irna H.N. Hadi. (1985). Soewardi Soerjaningrat dalam Pengasingan. Jakarta: Balai Pustaka. Sugiarta, I.M.,Mardana.I.B.P, Adiarta, A.,&Artanayasa, I.W. (2019). Filsafat Pendidikan Ki Hadjar Dewantara (Tokoh Timur). Jurnal Filsafat Indonesia. Vol 2 No 3Tahun 2019 h.124-136. Tauchid, Muchammad. (2011). Perjuangan dan Ajaran Hidup Ki Hadjar Dewantara. Yogyakarta: Majelis Luhur Tamansiswa Yogyakarta. Widodo, Bambang. (2017). Biografi: Dari Suwardi Suryaningrat Sampai Ki Hadjar Dewantara. Jakarta: Makalah Seminar “Perjuangan Ki Hadjar Dewantara dari Politik ke Pendidikan. 2840
https://openalex.org/W4225731513	https://www.duo.uio.no/bitstream/10852/100151/1/1-s2.0-S0048969722019830-main.pdf	English	null	Spatiotemporal distribution of antimicrobial resistant organisms in different water environments in urban and rural settings of Bangladesh	Science of the total environment	2,022	cc-by	12,532	G R A P H I C A L A B S T R A C T G R A P H I C A L A B S T R A C T • Antibiotic resistant bacteria (ARB) pose a major risk to water quality. • ARB concentrations in aquatic environ- ments in Bangladesh are high. • Wastewater from urban markets and river water is highly contaminated with ARB. • Seasonal differences in the concentration of major ARB were not signiﬁcant. • GIS based maps are useful for tracing envi- ronmental transmission of ARB. • Antibiotic resistant bacteria (ARB) pose a major risk to water quality. • ARB concentrations in aquatic environ- ments in Bangladesh are high. • Wastewater from urban markets and river water is highly contaminated with ARB. • Seasonal differences in the concentration of major ARB were not signiﬁcant. • GIS based maps are useful for tracing envi- ronmental transmission of ARB. • Antibiotic resistant bacteria (ARB) pose a major risk to water quality. • Antibiotic resistant bacteria (ARB) pose a major risk to water quality. • ARB concentrations in aquatic environ- ments in Bangladesh are high. • Wastewater from urban markets and river water is highly contaminated with ARB. A R T I C L E I N F O Editor: Warish Ahmed Keywords: Antimicrobial resistance Geographical information system (GIS) Spatial mapping Aquatic environment ESBL E. coli Water contamination journal homepage: www.elsevier.com/locate/scitotenv journal homepage: www.elsevier.com/locate/scitotenv Contents lists available at ScienceDirect Contents lists available at ScienceDirect Spatiotemporal distribution of antimicrobial resistant organisms in different water environments in urban and rural settings of Bangladesh Muhammad Asaduzzaman a,b,⁎, Emily Rousham c, Leanne Unicomb d, Md. Rayhanul Islam b, Mohammed Badrul Amin b, Mahdia Rahman b, Muhammed Iqbal Hossain b, Zahid Hayat Mahmud b, Mark Szegner e, Paul Wood e, Mohammad Aminul Islam b,f,⁎⁎ a Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway b Food Safety and One Health Laboratory, Laboratory Sciences and Services Division, International Centre for Diarrhoeal Disease Research, Bangla c Centre for Global Health and Human Development, School of Sport, Exercise and Health Sciences, Loughborough University, UK d Environmental Intervention Unit, Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka e Geography and Environment, School of Social Sciences and Humanities, Loughborough University, Loughborough, UK f Paul G. Allen School for Global Health, Washington State University, Pullman, WA, USA a Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Oslo, Norway b Food Safety and One Health Laboratory, Laboratory Sciences and Services Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka 1212, Bangladesh c Centre for Global Health and Human Development, School of Sport, Exercise and Health Sciences, Loughborough University, UK d Environmental Intervention Unit, Infectious Diseases Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), Dhaka 1212, Bangladesh e Geography and Environment, School of Social Sciences and Humanities, Loughborough University, Loughborough, UK f Paul G. Allen School for Global Health, Washington State University, Pullman, WA, USA ⁎ Correspondence to: M. Asaduzzaman, Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Kirkeveien 166, Frederik Holsts hus, 0450 Oslo, Norway. ⁎⁎ Correspondence to: M.A. Islam, Paul G. Allen School for Global Health, Washington State University, 240 SE Ott Road, Pullman, WA 99164, USA. E-mail addresses: muhammad.asaduzzaman@medisin.uio.no (M. Asaduzzaman), amin.islam@wsu.edu (M.A. Islam). Science of the Total Environment 831 (2022) 154890 Science of the Total Environment 831 (2022) 154890 2.1. Study design Our primary objective was to adopt an integrated spatial approach to es- timate the prevalence and abundance of antimicrobial resistant organisms and antimicrobial resistant genes in different environmental compartments that are likely to be impacted by intensive poultry production/selling prac- tices in both urban and rural areas of Bangladesh. We investigated the point prevalence of ESBL-Ec and CR-Ec and associated resistant genes in water samples collected from drinking water supply, wastewater drains and sur- face water bodies in three different settings in Bangladesh. Sites included urban food markets in high-density residential areas in Dhaka city; rural small-scale commercial poultry farms, and rural households in Mirzapur sub-district (upazila) in Tangail district. In urban areas, we selected food markets as these are the largest outlets of commercial poultry where birds are slaughtered and processed on site with no regulated waste disposal sys- tem. Fecal wastes produced within urban markets are mostly disposed into the municipal drainage system through direct washout. Due to the com- plexity of the wastewater circulation and disposal system, we only sampled wastewater from markets at the points of discharge to the municipal drain- age system. Although there are rivers surrounding Dhaka city, those are not in proximity of the selected markets and therefore were not sampled as there was no spatial connection. A signiﬁcant proportion of deaths from AMR occur in low and middle- income countries (LMICs) where AMR rates are much higher than high- income countries (Murray et al., 2022). Community carriage of extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) is increasing at an estimated annual global growth rate of 1.5% with the highest preva- lence reported from Southeast Asia (Bezabih et al., 2021). In our previous study we found that 74% of healthy infants in rural Bangladesh were colo- nized with ESBL- Escherichia coli (ESBL-Ec) (Islam et al., 2019). Factors that contribute to AMR carriage in LMICs include widespread availability and frequent use of antibiotics (Ghafur, 2010), high population density, and limited access to safe water and sanitation (Laxminarayan et al., 2013; Woerther et al., 2013). Globally, 1.8 billion people drink fecal- contaminated water, while Africa and Southeast Asia have the highest prev- alences of water contamination (Bain et al., 2014). More than 80% of sew- age or wastewater generated in LMICs ﬂows into the environment untreated (UN Water, 2018; DeFrancis, 2011). A B S T R A C T The aim of the current study was to adopt an integrated spatial approach to quantify the prevalence and abundance of AMR organisms and ARGs in different environmental com- partments in both urban and rural areas of Bangladesh and visualize the re- sults using geographic information system (GIS). 1. Introduction Antimicrobial resistance (AMR) is a global health problem affecting all humans, animals and environments (Lanyon et al., 2021). The role of the environment as an important reservoir for transmission of AMR to both humans and animals is increasingly recognized on a global scale with grow- ing concerns regarding the public health threat (Fletcher, 2015). The 2017 United Nations Environment Programme report highlighted that the envi- ronment is key to AMR (Gaze and Depledge, 2017). Although AMR is a nat- ural phenomenon of bacteria, the increased use of antimicrobial compounds in human, animal and agricultural sectors and their subsequent release into the environment has promoted the emergence of novel resistance and their dissemination in wider spaces (D'Costa et al., 2011; Holmes et al., 2016). Up to 90% of administered antibiotics are released in an active form through urine and feces from treated patients and animals (Singer et al., 2016), of which a signiﬁcant proportion passes directly to the environment (Levison and Levison, 2009). Although the concentration of antibiotics in the environment is much less than the therapeutic doses, these are sufﬁcient to induce antimicrobial resistance in environmental microbiota (Gullberg et al., 2011; Lundström et al., 2016; Kraupner et al., 2018). Compounding con- cerns are the release of many other compounds in the environment which can co-select resistance to antibiotics (Baker-Austin et al., 2006; Seiler and Berendonk, 2012; Wales and Davies, 2015). Consequently, antibiotic resistant organisms are ubiquitously found in environmental compartments (Holmes et al., 2016). Moreover, there is evidence that environmental bacteria share antibiotic-resistant genes (ARGs) with clinically important bacteria or patho- genic bacteria via horizontal gene transfer (Cantón et al., 2012; Ashbolt et al., 2013), and that humans with high exposure to contaminated environ- ment are more likely to be colonized by antibiotic resistant organisms com- pared to people with less exposure (Leonard et al., 2018). 2.1. Study design In addition, pharmaceutical waste containing high concentrations of antibiotics; hospital efﬂuent (Rozman et al., 2020; Zhang et al., 2020) and agricultural run-off from an- imal husbandry carrying antibiotic resistant organisms are directly released to the environment (Lübbert et al., 2017). All these wastes contain a cock- tail of microorganisms of human, animal and environmental origin and res- idues of antibiotics/antimicrobial agents, which can select for AMR. In rural communities (households and farms), we sampled ground water (drinking water from deep tubewells) and the wastewater outlets ad- jacent to individual households or poultry farms. We then sampled pond water located within the same property, and river water at the closest point to the property. Ponds in rural areas are found on individual proper- ties (homesteads) and are often artiﬁcially constructed (known as ‘pukur’ in Bengali, ranging from 150 to 1000 square metres area) (Huq, 2017). The ponds collect run off from household drains, or drainage ditches as well as rainwater/surface water. They are used for a multitude of purposes, in- cluding waste disposal, bathing of people and animals, cooking and wash- ing utensils. For the urban food markets in Dhaka, we sampled the municipal water supply and the wastewater directly discharged from mar- ket stalls consisting mainly of market run-off. The aquatic environment is very important in the context of AMR, espe- cially for a country like Bangladesh. Bangladesh has a landscape populated with numerous ponds and wetlands along with more than 250 rivers which are an integral part of human and animal subsistence (Islam, 2016). How- ever, studies reporting contamination of surface waterbodies with antibiotic resistant bacteria (ARB) in Bangladesh are limited (Haque et al., 2014; A B S T R A C T Science of the Total Environment 831 (2022) 154890 Science of the Total Environment 831 (2022) 154890 Ec, CR-Ec, blaCTXM-1 and blaNDM-1 and these were signiﬁcantly higher in urban compared to rural samples (p < 0.05). ESBL-Ec is ubiquitous in drinking water, wastewater and surface water bodies in both rural and urban areas of Bangladesh. CR-Ec is less widespread but found at a high prevalence in wastewater discharged from urban food mar- kets and in rural river samples. Surveillance and monitoring of antibiotic resistant organisms and genes in waterbodies is an important ﬁrst step in addressing environmental dimensions of AMR. Kamruzzaman et al., 2013) and none has investigated seasonal or spatial variation. A study conducted in urban Dhaka showed 71% and 62% of environmental water samples were positive for the ESBL gene, blaCTX-M-15, and the carbapenemase gene, blaNDM-1, respectively (Toleman et al., 2015). Similarly, wastewater samples in Dhaka city particularly at sites adjacent to hospitals were found to be contaminated with multi-drug resistant organisms including blaNDM-1 positive E. coli (Rabbani et al., 2017). While aquatic envi- ronments are likely to play a major role in the transmission of AMR, identiﬁ- cation of major aquatic reservoirs to which humans and animals are exposed is the key to successful implementation of community-based intervention strategies for combating AMR (Singer et al., 2016). The aim of the current study was to adopt an integrated spatial approach to quantify the prevalence and abundance of AMR organisms and ARGs in different environmental com- partments in both urban and rural areas of Bangladesh and visualize the re- sults using geographic information system (GIS). Kamruzzaman et al., 2013) and none has investigated seasonal or spatial variation. A study conducted in urban Dhaka showed 71% and 62% of environmental water samples were positive for the ESBL gene, blaCTX-M-15, and the carbapenemase gene, blaNDM-1, respectively (Toleman et al., 2015). Similarly, wastewater samples in Dhaka city particularly at sites adjacent to hospitals were found to be contaminated with multi-drug resistant organisms including blaNDM-1 positive E. coli (Rabbani et al., 2017). While aquatic envi- ronments are likely to play a major role in the transmission of AMR, identiﬁ- cation of major aquatic reservoirs to which humans and animals are exposed is the key to successful implementation of community-based intervention strategies for combating AMR (Singer et al., 2016). A B S T R A C T The spatial distribution of clinically important antibiotic resistant bacteria (ARB) and associated genes is important to identify the environmental distribution of contamination and ‘hotspots’ of antimicrobial resistance (AMR). We con- ducted an integrated survey of AMR in drinking water, wastewater and surface water (rivers and ponds) in three set- tings in Bangladesh: rural households, rural poultry farms, and urban food markets. Spatial mapping was conducted via geographic information system (GIS) using ArcGIS software. Samples (n = 397) were analyzed for the presence of extended-spectrum β-lactamase-producing Escherichia coli (ESBL-Ec), carbapenem-resistant E. coli (CR-Ec) and resis- tance genes (blaCTX-M-1, blaNDM-1). In rural households, 5% of drinking water supply samples tested positive for ESBL- Ec, and a high proportion of wastewater, pond and river water samples were positive for ESBL-Ec (90%, 76%, and 85%, respectively). In poultry farms, 10% of drinking water samples tested positive for ESBL-Ec compared to a high prevalence in wastewater, pond and river water (90%, 68%, and 85%, respectively). CR-Ec prevalence in household wastewater and pond water was relatively low (8% and 5%, respectively) compared to river water (33%). In urban areas, 38% of drinking water samples and 98% of wastewater samples from food markets tested positive for ESBL- Ec while 30% of wastewater samples tested positive for CR-Ec. Wastewaters had the highest concentrations of ESBL- ⁎ Correspondence to: M. Asaduzzaman, Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, University of Oslo, Kirkeveien 166, Frederik Holsts hus, 0450 Oslo, Norway. ⁎⁎ Correspondence to: M.A. Islam, Paul G. Allen School for Global Health, Washington State University, 240 SE Ott Road, Pullman, WA 99164, USA. E-mail addresses: muhammad.asaduzzaman@medisin.uio.no (M. Asaduzzaman), amin.islam@wsu.edu (M.A. Islam). orrespondence to: M. Asaduzzaman, Department of Community Medicine and Global Health, Institute of Health and Society, Faculty of Medicine, Uni s hus, 0450 Oslo, Norway. orrespondence to: M.A. Islam, Paul G. Allen School for Global Health, Washington State University, 240 SE Ott Road, Pullman, WA 99164, USA. -mail addresses: muhammad.asaduzzaman@medisin.uio.no (M. Asaduzzaman), amin.islam@wsu.edu (M.A. Islam). http://dx.doi.org/10.1016/j.scitotenv.2022.154890 Received 23 November 2021; Received in revised form 28 February 2022; Accepted 24 March 2022 Available online 29 March 2022 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 0048-9697/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://crea M. Asaduzzaman et al. 2.2. Study settings and duration We collected environmental samples over two periods from February to April (which we considered as winter) (n = 197) and August to November 2 M. Asaduzzaman et al. Science of the Total Environment 831 (2022) 154890 8 °C) immediately after collection and transported to the laboratory within 8 h of collection. Samples were processed immediately upon arrival in the lab. (summer) (n = 200). Average air temperatures in Bangladesh range from 17°−20.6 °C in winter and 26.9°−31.1 °C in summer (Shahid, 2010). Lo- cally, winter is considered as the dry season due to low rainfall and limited navigability of water bodies and summer is considered as the wet season. Different households, farms and markets within the area were included in each sampling period to cover most of the aquatic environments in the study area. 2.3.3. Temperature, pH and conductivity assessment of water samples 2.3.3. Temperature, pH and conductivity assessment of water samples Temperature and electrical conductivity of water samples were assessed using a total dissolved solids (EC-TDS) meter (Hanna Instruments, UK; Model: HI 98311). Water pH was measured with an accuracy of ±0.1 using a portable pH meter (Hanna Instruments, UK; Model: HI 98127). The probe/electrode of both the EC-TDS meter and pH meter were sub- merged in the water samples at the point of collection. All readings were taken following manufacturer's instructions. For assessment of ARB, we investigated ESBL-Ec and CR-Ec, and for ARGs, we considered blaCTX-M-1 and blaNDM-1, which are the most common genes found in ESBL-Ec and CR-Ec, respectively (Bevan et al., 2017; Nordmann and Poirel, 2014). WHO has also recommended ESBL-Ec as an indicator organism for integrated One Health surveillance of AMR due to its clinical signiﬁcance, ubiquitous presence in humans, animals and the en- vironment, and its ability to be easily transmitted between these compart- ments (WHO, 2020a). 2.3.4. Culture of samples for E. coli resistant to third generation cephalosporin or carbapenem For all water sample types, 3 × 100 ml of water was ﬁltered through three separate 0.22 μm nitrocellulose membrane (Sartorius Stedim Biotech GmbH, Goettingen, Germany) using a Millipore manifold ﬁltration system (EZ-Fit™Manifold, Merck KGaA, Darmstadt, Germany) in which microor- ganisms were retained on the membrane surface. Membrane ﬁlters were placed in an upright position on three mTEC agar plates (BD Difco®); one without supplementation, one supplemented with cefotaxime (1 mg/l) and the other one with meropenem (0·5 mg/l) (Rousham et al., 2021). 2.2. Study settings and duration For each batch of sample, the same volume of autoclaved distilled water passed through nitrocellulose membranes plated on the same culture media was considered as lab blanks. Plates were incubated at 35 ± 0.5 °C for 2 h followed by further incubation at 44.5 ± 0.2 °C for approximately 22–24 h. After incubation, magenta colour colonies, typical of E. coli, were counted. At least two isolated colonies were extracted from each sam- ple plate and stored in glycerol stocks at −80 °C. 2.3. Sampling strategy and laboratory analysis 2.3.5. Quantiﬁcation of blaNDM-1 and blaCTX-M-1 genes For all types of water samples, 100 ml of sample was ﬁltered through a 0.22 μm nitrocellulose membrane (Sartorius Stedim Biotech GmbH, Goettingen, Germany) using a Millipore manifold ﬁltration system (EZ- Fit™Manifold, Merck KGaA, Darmstadt, Germany). The ﬁlters were placed in 2-ml tubes with glass beads (GeneRite, North Brunswick, NJ) and DNA was extracted from the ﬁlters using the MO Bio Power Water DNA isolation Kit (MO BIO Laboratories Inc., USA) following user's instructions. DNA ex- traction was performed in sets of 10–20 ﬁlters at a time. For each extraction set, a process blank was created by passing through the same volume of autoclaved distilled water through nitrocellulose membrane and used for DNA extraction. Extraction process blanks were treated in the same way as the water samples, except that the extraction blank bead tube had no ﬁlter. 2.3.1. Sampling procedure In the rural site, 40 households and 40 poultry farms were selected from different villages in Mirzapur sub-district which is surrounded by ﬁve rivers and their branches namely Bangshi, Lauhojong, Longla, Dhaleswari and Futjani. All the selected villages were in the vicinity of river branches. From each village (n = 20), the data collector selected 2 households with backyard poultry, which were 10 households apart from each other. For the selection of farms, 1 broiler poultry farm per village was selected using convenience sampling. For each household or farm, we collected sam- ples from the drinking water supply to the individual household (tubewell water, 1 per household/farm, n = 80) located on the household/farm pre- mises, wastewater (in the vicinity of the household premises or farm waste- water drains, 1 per household/farm, n = 80), water from the closest pond to the property (1 per household/farm, n = 80) and river water (at the clos- est point, approximately 0.5 to 1.0 km from the household/farm, n = 80). For biochemical identiﬁcation and conﬁrmation, one E. coli isolate from each sample was tested using API20E kits (BioMérieux, France). For any sample where the ﬁrst isolate tested negative for E. coli, the second isolate was tested to conﬁrm E. coli identiﬁcation. All isolates obtained from cefo- taxime plates were tested for ESBL production by double disc synergy test (DDST) following the method described by CLSI (CLSI 2016). Isolates from meropenem plates were conﬁrmed as resistant to carbapenem by doing susceptibility test against meropenem (10 μg), imipenem (10 μg) and ertapenem (10 μg). In urban areas, we selected 40 different urban food markets within Dhaka North and South City Corporation following convenience sampling. In each market, the data collector selected the 11th eligible poultry stall starting from the center. Then, municipal water supply samples within the market (drinking water, n = 40, 1 per market) and wastewater drain samples containing market run-off (n = 40, 1 per market) were collected, both of which were near the poultry stall. We collected samples during two different seasons to characterize the potential temporal variation in ABR and ARGs with changes in season tem- perature, pH, conductivity, and surface water volume. The ﬁrst sampling took place in winter when surface water levels were low and the second sampling was conducted in summer. 2.3.5. Quantiﬁcation of blaNDM-1 and blaCTX-M-1 genes 2.5. Statistical analyses The prevalence of ESBL-Ec and CR-Ec was assessed as the proportion of positive culture results obtained from the total number of samples for a given environmental compartment. As E. coli counts displayed a non- normal distribution, all counts were log transformed to log10 (1 + x), where x is the bacterial count. In addition, all zero values (negative for bac- teria and quantitative gene counts) were assigned with a random generated number between zero and the limit of detection (LOD) for each sample type (drinking water, surface water and wastewater) (Canales et al., 2018). The copy number of blaNDM-1 and blaCTX-M-1 genes was estimated using qPCR. While the number of ESBL-Ec or CR-Ec provides an estimation of the culturable ESBL-producing E. coli or carbapenem resistant E. coli, copies of blaCTX-M-1 and blaNDM-1 genes provide a presumptive estimation of the total number of ESBL-producing and carbapenem resistant organisms pres- ent in the sample. CR-Ec was less common than ESBL-Ec and no drinking water samples from any of the three sites were positive for CR-Ec. However, in urban food markets, 30% of wastewater samples were positive for CR-Ec, which is signiﬁcantly higher than wastewater samples from rural households and farms (8% and 5%, respectively, p < 0.05, ANOVA) (Table 1). Around 33% of river water samples adjacent to rural households were positive for CR-Ec while only 13% of river water samples adjacent to farms were posi- tive (p < 0.05, Chi-square test) (Table 1). In pond water, the difference in prevalence of CR-Ec between household ponds and farm ponds was not sta- tistically signiﬁcant (5% versus 8% respectively, p = 0.470, Chi-square test) (Table 1). ESBL-Ec and CR-Ec counts were expressed as a proportion of total culturable E. coli counts per sample according to the recommendation by the WHO Tricycle project guideline (Matheu et al., 2017). For each sample, we calculated the proportion of resistant bacteria by dividing the number of colonies on culture media (mTEC) with (cefotaxime) vs. without antibiotic supplementation. Only ESBL-Ec or CR-Ec positive samples were used for calculating the proportions and for each sample type, median proportion and interquartile range (IQR) were enumerated. Frequencies of ARGs were expressed as a proportion of all samples that were positive for ESBL- Ec isolates. Fig. 1. Winter and summer prevalence of ESBL-Ec and CR-Ec in differnt aquatic environments CR-Ec = carbapenem resistant E. coli; ESBL-Ec = extended- spectrum β-lactamase-producing E. coli. 3.1. Prevalence of ESBL-Ec and CR-Ec in aquatic environmental samples In rural households, 5% of tubewell water (drinking water) samples tested positive for ESBL-Ec by direct culture of samples, while 90% of wastewater, 76% of pond water and 85% of river water samples were pos- itive for ESBL-Ec. Similarly, 10% of drinking water samples from poultry farms tested positive for ESBL-Ec while a higher prevalence was observed in wastewater, pond and river water samples (90%, 68%, and 85%, respec- tively) (Table 1, Fig. 1). In urban food markets, 38% of drinking water sam- ples and 98% of wastewater samples were positive for ESBL-Ec. Table 1 Table 1 Prevalence of ESBL-Ec, CR-Ec, blaCTX-M-1, and blaNDM-1 in water samples from rural households, poultry farms and urban food markets. Prevalence of ESBL-Ec, CR-Ec, blaCTX-M-1, and blaNDM-1 in water samples from rural households, poultry farms and urban food markets. Locations Organisms Drinking water n (%) Wastewater n (%) Pond water n (%) River water n (%) Rural households ESBL-Ec 2 (5) 36 (90) 28 (76) 34 (85) CR-Ec 0 (0) 3 (8) 2 (5) 13 (33) blaCTX-M-1 3 (8) 24 (60) 5 (14) 11 (28) blaNDM-1 2 (5) 14 (35) 3 (8) 8 (20) Poultry farms ESBL-Ec 4 (10) 36 (90) 27 (68) 34 (85) CR-Ec 0 (0) 2 (5) 3 (8) 5 (13) blaCTX-M-1 0 (0) 17 (43) 5 (13) 3 (7) blaNDM-1 0 (0) 13 (33) 2 (5) 6 (15) Urban food markets ESBL-Ec 15 (38) 39 (98) NA NA CR-Ec 0 (0) 12 (30) NA NA blaCTX-M-1 0 (0) 33 (83) NA NA blaNDM-1 0 (0) 23 (58) NA NA ESBL-Ec = extended-spectrum β-lactamase-producing E. coli. CR-Ec = carbapenem-resistant E. coli. NA = no surface water samples were collected in urban setting. 2.4. Geospatial mapping and geocoding GPS (geographic positioning system) coordinates of all households, farms and markets and the downstream sample collection sites were re- corded using a GPS device (Garmin, etrex 10). For household locations, GPS coordinates were recorded at the entrance to the kitchen, to represent the center of the homestead. Farm and urban market locations were recorded at the entrance of poultry sheds and the market stalls, respectively. GIS coordinates (northing and easting) were stored and downloaded to Microsoft Excel, recorded to three decimal places and ﬁnally converted to GIS shape ﬁle in ArcGIS software. Later, the GIS shape ﬁles were linked with microbiological and laboratory data. All the GIS coordinates related to water samples were geolocated on a base map using ArcMap® version 10.6.1 in ArcGIS (ESRI®, Redlands, CA). Each data point in the maps represents an environmental sample with the concentration of ESBL E. coli indicated by the size of the data point using three arbitrary cut-points for low (log101.0- log102.99 CFU/100 ml), middle (log10 3.0- log105.99 CFU/100 ml) and high (log106.0- log109.99 CFU/100 ml) concentrations. Black dots indicate a sample that was negative for ESBL-Ec. 2.3.2. Sample collection In rural households and farms, we collected drinking water samples directly from tubewells and in urban markets, we collected water samples directly from taps for municipal water supply. For tubewell samples, we op- erated the hand pump of the tubewell continuously for 5 min, followed by heating the mouth of the pump by using a gas torch and then again pumping out several litres of water. At this stage, we collected water sam- ples aseptically into a sterile bottle (Nalgene, Rochester, New York). For tap water samples, we ﬁrst cleaned the outside nozzle of the tap and allowed the water to run to waste for 1 min. Using a gas torch, we heated the nozzle and allowed the water to run to waste for a few seconds. We col- lected water samples aseptically into a sterile bottle (Nalgene, Rochester, New York) and replaced the cap carefully. We collected approximately 150 ml of water samples. Gene ampliﬁcation for blaNDM-1 and blaCTX-M-1 was carried out in a Bio- Rad CFX96 real-time PCR platform using TaqMan technology. Details of primers, probes and PCR conditions are provided in Supplementary mate- rial 1. A recombinant plasmid DNA (pUCIDT-Kanr) containing target gene sequences (blaCTX-M-1 and blaNDM-1) was commercially produced (IDT Inc.) and used to prepare a known concentration of DNA solution. The stock solution of plasmid DNA (40 ng/μl, 2.35 × 1010 copies/2 μl volume) was 10-fold serially diluted to make solutions containing 101–107 copies of plasmid DNA that were used for generating a standard curve. The ampliﬁ- cation of standard DNA was linear over dilutions (r2 = 0.999; slope = −3.66, y-int = 40.614, and E = 99.0%). The Cq value variation for blaCTX-M-1 and blaNDM-1 was 31.35–34.79 at the limit of detection (LOD). We collected wastewater samples from three different locations of the drain proximal to rural households, farms and urban food markets. The total volume of the pooled sample was approximately 900 ml (300 ml from each location). In the case of ponds and rivers, we collected water samples by dipping a sterile bottle into the water approximately 30 cm below the water surface. We placed all water samples in a cold box (4 °C– 3 Science of the Total Environment 831 (2022) 154890 M. Asaduzzaman et al. Each sample was run in triplicate, and ampliﬁcation was only considered as positive if all three technical replicates showed a positive result. 2.3.2. Sample collection Mean copy number of genes calculated from three replicates of each sample was used in subsequent analysis. Data were analyzed using Bio-Rad CFX Manager (version 3.1). 2.5. Statistical analyses We used IBM SPSS (version 23.0) and Stata (version 13.0) software for data analysis. ARB and ARG counts were tested for symmetric distribution using Shapiro-Wilk test. Most of the variables showed non-normal distribu- tions for which non-parametric statistical tests such as Wilcoxon test or Kruskal Wallis test were executed. The percent prevalence for ARB and ARGs was compared using Chi-square test. Binary logistic regression was used to identify the potential risk factors for ESBL-E. coli contamination of wastewater, and pond water. The p-value for the Pearson correlation coefﬁcient was calculated to ﬁnd out if contamination of surface water is as- sociated with physicochemical properties of water including pH, conductiv- ity, and temperature in different seasons. For multiple comparisons among different sample types, Tukey's post hoc test was employed. Statistical sig- niﬁcance was deﬁned by 95% conﬁdence intervals at p < 0.05. Fig. 1. Winter and summer prevalence of ESBL-Ec and CR-Ec in differnt aquatic environments CR-Ec = carbapenem resistant E. coli; ESBL-Ec = extended- spectrum β-lactamase-producing E. coli. 4 Science of the Total Environment 831 (2022) 154890 M. Asaduzzaman et al. Fig. 2. Concentrations of ESBL-Ec (log10 CFU/ml) in household drinking water supply, household wastewater, household pond water and downstream river water adjacent to each household for 40 rural households in Mirzapur. Each data point represents an environmental sample with the concentration of log10 ESBL-Ec indicated by the size of the data point using three arbitrary cut-points for low (log101.0- log102.99), middle (log10 3.0- log105.99) and high (log106.0- log109.99) concentrations. Black dots indicate a sample that was negative for ESBL-Ec. M. Asaduzzaman et al. Science of the Total Environment 831 (2022) 154890 Fig. 2. Concentrations of ESBL-Ec (log10 CFU/ml) in household drinking water supply, household wastewater, household pond water and downstream river water adjacent to each household for 40 rural households in Mirzapur. Each data point represents an environmental sample with the concentration of log10 ESBL-Ec indicated by the size of the data point using three arbitrary cut-points for low (log101.0- log102.99), middle (log10 3.0- log105.99) and high (log106.0- log109.99) concentrations. Black dots indicate a sample that was negative for ESBL-Ec Fig. 3. Concentrations of ESBL-Ec (log10 CFU/ml) in drinking water supply, farm wastewater, farm pond water and downstream river water adjacent to each farm for 40 poultry farms in Mirzapur. 3.2. Spatial distribution of ESBL-Ec and CR-Ec in aquatic environments 3.2. Spatial distribution of ESBL-Ec and CR-Ec in aquatic environments 3.2. Spatial distribution of ESBL-Ec and CR-Ec in aquatic environments Kruskal Wallis test) (Table 2; Fig. 4). The mean ESBL-Ec counts (log10 CFU/ 100 ml) in wastewater samples and drinking water samples were 5.59 ± 2.85 SD and 0.53 ± 0.34 SD respectively. The abundance of CR-Ec was also very high in market wastewater compared to all other samples with a mean CR-Ec of 3.48 ± 1.61 SD log10 CFU/100 ml. The ratio of ESBL-Ec to total number of E. coli colony forming units in drinking water samples from urban food markets were signiﬁcantly higher than that of wastewater (20.79% vs. 6.67%) (p < 0.001, Kruskal Wallis test). The spatial distribution of ESBL-Ec in aquatic samples is presented for rural households (Fig. 2), poultry farms (Fig. 3) and urban food markets in Dhaka city (Fig. 4). Spatial distribution clearly demonstrates the rela- tively lower concentration of ARB in ground water or water supply com- pared to wastewater. In rural households and farms, drinking water samples were mostly negative for ESBL-Ec while the majority of wastewa- ter, pond and river water samples were positive for these organisms. In urban markets, a higher proportion of drinking water samples were positive for ESBL-Ec compared to the samples from rural areas (Fig. 4). p We also compared the concentration of ARB and ARGs in rural and urban settings (Table 3) which showed greater contamination in urban en- vironments. The mean ESBL-Ec counts (log10 CFU/100 ml) in wastewater adjacent to urban settings (5.59 ± 2.85 SD) were signiﬁcantly higher than those counts in rural settings (2.99 ± 2.39 SD) (p < 0.001, Wilcoxon Rank Sum test) (Table 3). Similarly, drinking water adjacent to rural and urban settings had mean ESBL-Ec counts (log10 CFU/100 ml) of 0.23 ± 0.16 SD and 0.53 ± 0.34 SD, respectively (p < 0.05, Wilcoxon Rank Sum test). The mean total E. coli counts (log10 CFU/100 ml) was signiﬁcantly higher in urban wastewater (6.91 ± 1.25 SD) and rural drinking water (0.93 ± 0.99 SD) (p < 0.001, Wilcoxon Rank Sum test) which is consistent with the result of the median proportion of ESBL-Ec in total E. coli (Table 3). 3.2. Spatial distribution of ESBL-Ec and CR-Ec in aquatic environments CR-Ec mean counts (log10 CFU/100 ml), concentration of blaCTX-M-1, and blaNDM-1 genes (log10 gene copy number/100 ml) in wastewater were sig- niﬁcantly higher in the urban markets compared to those from rural areas (p < 0.05, Wilcoxon Rank Sum test). We found a signiﬁcantly higher concentration of ESBL-Ec and CR-Ec in wastewater compared to pond and river water samples (p < 0.001, Kruskal Wallis test) except for CR-Ec in poultry farm wastewater (Table 2; Figs. 2 and 3). The mean ESBL-Ec counts (log10 CFU/100 ml) in wastewater adja- cent to rural households and farms were 2.91 ± 1.32 SD and 3.06 ± 1.55 SD, respectively (Table 2). The pond and river water adjacent to rural households had mean ESBL-Ec counts (log10 CFU/100 ml) of 1.49 ± 1.10 SD and 1.66 ± 1.08 SD, respectively. In pond and river water adjacent to farms, the mean ESBL-Ec counts (log10 CFU/100 ml) were 1.16 ± 0.95 SD and 1.38 ± 0.82 SD, respectively. River water samples appeared to have a signiﬁcantly higher proportion of ESBL-Ec over total E. coli in the sample compared to pond and wastewater samples in rural settings (Table 2). We also found a relatively high concentration of CR-Ec in the wastewater from rural households and farms with a mean count of 2.07 ± 1.19 SD and 1.70 ± 0.38 SD log10 CFU/100 ml, respectively. The concentration of CR-Ec in pond water samples adjacent to rural households and farms were higher (1.13 ± 0.40 SD, 0.69 ± 0.37 SD, respectively) than the river water samples adjacent to rural households and farms (0.85 ± 0.44 SD, 0.55 ± 0.22 SD, respectively). Overall, river water samples had a higher concentration of ESBL-Ec than pond water samples but in the case of CR-Ec this association was reversed. We investigated the risk factors associated with AMR contamination, particularly in relation to waste disposal practices in poultry farms and rural households (Tables S1 & S2). To assess the risk of ESBL-Ec contamina- tion of wastewater and pond water in rural settings, we selected several var- iables such as number of poultry, types of waste disposed, distance of disposal sites and pond from households or farms, likelihood of pond water contamination with ESBL-Ec if wastewater of adjacent households or farms is positive for ESBL-Ec. M. Asaduzzaman et al. 2.5. Statistical analyses Each data point represents an environmental sample with the concentration of log10 ESBL-Ec indicated by the size of the data point using three arbitrary cut- points for low (log101.0- log102.99), middle (log10 3.0- log105.99) and high (log106.0- log109.99) concentrations. Black dots indicate a sample that was negative for ESBL-Ec. Fig. 3. Concentrations of ESBL-Ec (log10 CFU/ml) in drinking water supply, farm wastewater, farm pond water and downstream river water adjacent to each farm for 40 poultry farms in Mirzapur. Each data point represents an environmental sample with the concentration of log10 ESBL-Ec indicated by the size of the data point using three arbitrary cut- points for low (log101.0- log102.99), middle (log10 3.0- log105.99) and high (log106.0- log109.99) concentrations. Black dots indicate a sample that was negative for ESBL-Ec. 5 M. Asaduzzaman et al. Science of the Total Environment 831 (2022) 154890 3.4. Seasonal variation in physiochemical properties and prevalence of ESBL-Ec and CR-Ec in the aquatic environments 3.4. Seasonal variation in physiochemical properties and prevalence of ESBL-Ec and CR-Ec in the aquatic environments The average temperature of surface water bodies varied between the winter and summer sampling periods. In pond water, the mean temperature increased from 24.9 °C ± 1.80 SD in winter to 28.7 °C ± 2.40 SD in sum- mer (p < 0.001, t-test). River water temperature also increased signiﬁ- cantly from 25.4 °C ± 0.23 SD in winter to 29.0 °C ± 2.06 SD in summer (p < 0.001, t-test). We also observed signiﬁcant variations in water conductivity between seasons. The average conductivity of pond water in winter was 241.14 μS/cm ± 270.73 SD which was signiﬁcantly higher than in sum- mer, coinciding with monsoon (122.58 μS/cm ± 56.11 SD) (p < 0.05, t- test). Similarly, pond water pH was 7.80 ± 0.38 SD in winter, signiﬁcantly higher than that in summer (7.52 ± 0.36 SD), (p < 0.005, t-test). In river water, conductivity was signiﬁcantly higher in winter (245.80 ± 79.08 SD) than in summer (100.75 ± 38.40 SD) (p < 0.001, t-test) though no sig- niﬁcant change in pH (7.6 in both seasons) was observed. In the case of wastewater, conductivity was also higher in winter (782.54 ± 128.21 SD) than in summer (704.95 ± 94.71 SD) but the difference was not statis- tically signiﬁcant. There was a positive correlation between ESBL-Ec count (log10 CFU/ 100 ml) and conductivity of wastewater, pond and river water samples (r = 0.31; r = 0.49; r = 0.25 respectively, all p < 0.05, Pearson correla- tion). However, no correlation was observed between ESBL-Ec counts and water temperature or pH. The prevalence of ESBL-Ec in wastewater samples did not vary by season (all sites combined: 95% in winter and 90% in sum- mer, unadjusted OR 0.47, 95% CI 0.11, 1.99). The same was observed for CR-Ec in wastewater (all sites 11.7% vs 16.7% in winter and summer re- spectively, unadjusted OR 1.51, 95% CI 0.53, 4.29). Similarly, we did not ﬁnd any seasonal variations in the prevalence of ESBL-Ec in pond water samples (unadjusted OR 1.11, 95% CI 0.41, 3.00) or river water samples (unadjusted OR 0.67, 95% CI 0.19, 2.33), even after adjusting for study sites (Fig. 1). We observed the same for CR-Ec in pond water. 3.2. Spatial distribution of ESBL-Ec and CR-Ec in aquatic environments However, we did not ﬁnd any statistically signiﬁcant risk factors associated with ESBL-Ec contamination of wastewa- ter and pond water in rural settings. In urban food markets, wastewater had a signiﬁcantly higher concentra- tion of ESBL-Ec and CR-Ec compared to drinking water samples (p < 0.001, Fig. 4. Concentrations of ESBL-Ec (log10 CFU/ml) in market drinking water supplies and wastewater from 40 urban food markets within Dhaka city. Each data point represents an environmental sample with the concentration of log10 ESBL-Ec indicated by the size of the data point using three arbitrary cut-points for low (log101.0- log102.99), middle (log10 3.0- log105.99) and high (log106.0- log109.99) concentrations. Black dots indicate a sample that was negative for ESBL-Ec. Fig. 4. Concentrations of ESBL-Ec (log10 CFU/ml) in market drinking water supplies and wastewater from 40 urban food markets within Dhaka city. Each data point represents an environmental sample with the concentration of log10 ESBL-Ec indicated by the size of the data point using three arbitrary cut-points for low (log101.0- log102.99), middle (log10 3.0- log105.99) and high (log106.0- log109.99) concentrations. Black dots indicate a sample that was negative for ESBL-Ec. 6 M. Asaduzzaman et al. Science of the Total Environment 831 (2022) 154890 Table 2 Comparison of the abundance of ESBL-Ec, total E. coli, ESBL gene (blaCTX-M-1), CR-Ec and CR gene (blaNDM-1) in aquatic samples from rural households, poultry farms and urban food markets. Attributes Rural households Poultry farms Urban food markets Wastewater Ponds River pb Wastewater Ponds River pb Drinking water Wastewater pb (N = 40) (N = 37) (N = 40) (N = 40) (N = 40) (N = 40) (N = 40) (N = 40) Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD ESBL-E. coli counts (log10 CFU/100 ml) 2.91 ± 1.32a 1.49 ± 1.10a 1.66 ± 1.08a <0.001 3.06 ± 1.55a 1.16 ± 0.95a 1.38 ± 0.82a <0.001 0.53 ± 0.34 5.59 ± 2.85 <0.001 Total E. coli counts (log10 CFU/100 ml) 4.76 ± 0.93a 3.07 ± 1.22a 2.94 ± 1.02a <0.001 5.31 ± 1.38 2.94 ± 1.14a 2.70 ± 0.97 <0.001 0.93 ± 0.99 6.91 ± 1.25 <0.001 Median proportion (IQR) of ESBL-Ec in total Ec (%) 3.10 (17.93) 4.07 (10.34) 6.38 (22.34) 0.036 0.95 (5.12) 2.86 (6.05) 8.05 (20.93) 0.005 20.79 (29.01) 6.67 (20.97) <0.001 Carbapenem-resistant E. 3.2. Spatial distribution of ESBL-Ec and CR-Ec in aquatic environments coli (log10 CFU/100 ml) 2.07 ± 1.19 1.13 ± 0.40 0.85 ± 0.44 <0.001 1.70 ± 0.38 0.69 ± 0.37 0.55 ± 0.22 0.508 – 3.48 ± 1.61 – Median proportion (IQR) of Carbapenem Ec in total Ec (%) 0.02 (0.96) 0.02 (0.02) 0.70 (3.13) 0.038 0.01 (0.004) 0.29 (2.12) 0.91 (0.90) 0.235 – 0.02 (0.41) – blaCTX-M-1 (log10 gene copy number/100 ml) 4.35 ± 1.48a 0.75 ± 1.93a 1.38 ± 2.47a <0.001 4.04 ± 1.61a 0.68 ± 1.87a 0.41 ± 1.51a <0.001 2.81 ± 0.31 6.01 ± 1.85 <0.001 blaNDM-1 (log10 gene copy number/100 ml) 2.10 ± 3.61a 0.42 ± 1.46a 1.14 ± 2.71a 0.110 1.86 ± 2.83a 0.27 ± 1.16a 0.90 ± 2.69a 0.082 – 3.33 ± 3.21 – Using Tukey post-hoc comparisons a denotes pairs of groups signiﬁcantly different at p < 0.001; b denotes p-values generated using ANOVA/Kruskal Wallis test. 3.3. Distribution of blaCTX-M-1 and blaNDM-1 genes in aquatic environments The mean concentration of blaCTX-M-1 in drinking water samples from rural households was 2.81 ± 0.31 SD log10 gene copy number/100 ml. The concentration of blaCTX-M-1 genes in wastewater from rural households and farms was similar with a mean of 4.35 ± 1.48 and 4.04 ± 1.61 log10 copy number/100 ml of water, respectively. The average number of blaCTX-M-1 genes in pond water samples from rural households and farms was 0.75 ± 1.93 SD and 0.68 ± 1.87 SD, respectively, while the number was relatively higher in river water samples (1.38 ± 2.47 SD rural house- holds vs 0.41 ± 1.51 SD farms). The drinking water samples from the study sites were mostly negative for blaNDM-1 genes except two samples from rural households with a mean count of 1.74 ± 1.09 SD log10 gene copy number/100 ml. Wastewater samples from all three sites were posi- tive for blaNDM-1 with a higher concentration in urban markets (mean 3.33 ± 3.21 SD log10 gene copy number/100 ml) compared to rural house- holds (mean 2.10 ± 3.61 SD log10 gene copy number/100 ml) and farms samples (mean 1.86 ± 2.83 SD log10 gene copy number/100 ml) with a non-signiﬁcant statistical difference (p = 0.082, ANOVA test) (Table 2). However, the concentration of blaNDM-1 genes in river water samples adja- cent to rural households and farms were higher than that of pond water samples but the difference was not statistically signiﬁcant (Table 2). 4. Discussion Our study presents the spatial distribution of ARB and ARGs in drinking water, wastewater and surface water in rural and urban settings in Bangladesh. The high abundance of ESBL-Ec in wastewater within all set- tings, and drinking water in the urban area, raise important public health concerns. These ﬁndings also help to ﬁll highlighted gaps in research on the environmental dimensions of AMR (Larsson et al., 2018). Despite a greater awareness of environmental AMR, this has not yet been prioritized within environmental health policies or country-level national action plans on AMR, especially in LMICs (Rousham et al., 2018; WHO, 2020b). Interna- tional regulatory documents on water quality, such as WHO guidelines for drinking-water quality and WHO guidelines for the safe use of wastewater, excreta and greywater, do not provide guidelines on acceptable limits for antibiotic residues or clinically signiﬁcant MDR bacteria such as ESBL-Ec or CR-Ec in water bodies (WHO, 2017; WHO, 2006). This contributes to the lack of imperative for action on high AMR contamination as observed in this study. The visualization of concentration gradients through spatial mapping of resistant organisms from drinking water sources to wastewater run-off (urban areas) and to surface water bodies (rural areas) highlights environmental hotspots for AMR and the critical role of the aquatic envi- ronment as reservoirs for ARB and ARGs. These are likely to pose risks for onward transmission of AMR to humans and animals through direct and in- direct exposures. These ﬁndings also reinforce the need to evaluate poultry farming practices in Bangladesh which play a signiﬁcant role in the emer- gence of AMR (Rousham et al., 2018). The prevalence of ESBL-Ec in drinking water sampled directly from groundwater in rural households and farms was relatively low (5–10%), corresponding to other studies conducted in Bangladesh and India (Mahmud et al., 2020; Mahmud et al., 2019; Varghese and Roymon, 2013). Conversely, the high prevalence of ESBL-Ec in municipal supply water samples in urban markets (38%) is alarming as this same water sup- ply also serves residential areas in the city. We found that the estimated pro- portion of ESBL-Ec in total E. coli in municipal supply water samples was signiﬁcantly higher than that of wastewater. This might be due to presence of lower number of E. coli in municipal supply water compared to the waste- water which harbour E. coli from all different sources including both fecal and non-fecal origins. 4. Discussion This also indicates that people are directly exposed to a substantial number of ESBL-producing organisms by drinking contam- inated water on a regular basis and are thus likely to be colonized by these organisms. A previous study showed that around 80% of the municipal sup- ply water in Dhaka contain coliform bacteria, 63% of water samples were contaminated with E. coli and almost half of these E. coli (49%) were MDR pathogens with 9% ESBL-Ec (Talukdar et al., 2013). The increasing prevalence of ESBL-Ec in the municipal supply water in Dhaka city over the last decade is a serious concern that requires rapid attention by the re- spective authorities. Bangladesh is a densely populated country. Limited water treatment fa- cilities mean that only 17% of wastewater is treated, mostly in urban areas (Connor et al., 2017). In rural areas, wastewater often directly drains into the aquatic ecosystem via open channels into nearby ponds and rivers. We have demonstrated that wastewaters discharged from urban markets ﬂow into surrounding drains and ultimately the storm water carries a high abundance of ESBL-Ec, CR-Ec and associated resistance genes. In rural households and urban food markets in Bangladesh, wastewater gener- ally carries a combination of human, animal and general waste with the likelihood of both human and animal fecal contamination (Pickering et al., 2018; Huda et al., 2018; Harris et al., 2018; Harris et al., 2016) along with low concentrations of antibiotic residues (Karkman et al., 2017). The high prevalence of ESBL-Ec in wastewater discharged from the urban food markets, with more than 95% of samples positive, and a high mean concentration of ESBL-Ec (5.59 log10 CFU/100 ml) is notable. Of even greater concern is the high prevalence (30%) of CR-Ec in The occurrence of MDR organisms, resistance genes, and antibiotic res- idues in surface water bodies is well established outside Bangladesh, among similar settings in Asia (O'Flaherty and Cummins, 2017; Qiao et al., 2018; Waseem et al., 2018; Zhang et al., 2019; Singh et al., 2019; Reddy and Dubey, 2019; Ahammad et al., 2014). Natural ﬂow of untreated wastewater into surface water bodies including rivers, lakes or canals is commonly ob- served in many LMICs and is likely a key driver of environmental transmis- sion of clinically important antibiotic resistant organisms (Rabbani et al., 2017; Karkman et al., 2017; Islam et al., 2017). 3.4. Seasonal variation in physiochemical properties and prevalence of ESBL-Ec and CR-Ec in the aquatic environments However, the prevalence of CR-Ec in river water samples was signiﬁcantly lower in winter compared to summer (12.5% vs. 32.5%, Chi-square, p < 0.05 for household and farm river samples combined). The concentrations of total E. coli and ESBL-Ec (CFU/100 ml) in each study setting according to season are presented in Fig. 5 for rural households, farms and urban food markets. In rural households, the concentration of E. coli and ESBL-Ec was highest in wastewater and lowest in drinking water in both seasons. This trend was similar for all three settings. The level of contamination with E. coli and ESBL-Ec in pond and river water samples adjacent to poultry farms was 7 Science of the Total Environment 831 (2022) 154890 Table 3 Comparison of the abundance of ESBL-Ec, total E. coli, ESBL gene (blaCTX-M-1), CR-Ec and CR gene (blaNDM-1) in aquatic samples from rural and urban settings. Attributes Wastewater Drinking water Rural households + rural farms Urban food markets pb Rural households + rural farms Urban food markets pb (N = 80) (N = 40) (N = 80) (N = 40) Mean ± SD Mean ± SD Mean ± SD Mean ± SD ESBL-E. coli counts (log10 CFU/100 ml) 2.99 ± 2.39a 5.59 ± 2.85 <0.001 0.23 ± 0.16 0.53 ± 0.34 <0.05 Total E. coli counts (log10 CFU/100 ml) 5.03 ± 1.21a 6.91 ± 1.25 <0.001 0.93 ± 0.99 0.62 ± 0.65 <0.05 Median proportion (IQR) of ESBL-Ec in total Ec (%)* 2.01 (10.27) 6.67 (20.97) <0.05 33.73 (52.02) 20.79 (29.01) 0.410 Carbapenem-resistant E. coli (log10 CFU/100 ml) 1.91 ± 1.23 3.48 ± 1.61 <0.05 – – Median proportion (IQR) of Carbapenem Ec in total Ec (%)* 0.01 (0.01) 0.02 (0.41) 0.916 – – blaCTX-M-1 (log10 gene copy number/100 ml) 2.89 ± 1.25 6.01 ± 1.85 <0.001 4.12 ± 1.66 2.81 ± 0.31 0.697 blaNDM-1 (log10 gene copy number/100 ml) 1.98 ± 3.18 3.33 ± 3.21 <0.05 – – a denotes pairs of groups signiﬁcantly different at p < 0.001; *based on only positive isolates; b denotes p-values generated using Wilcoxon Rank Sum test. Table 3 Comparison of the abundance of ESBL-Ec, total E. coli, ESBL gene (blaCTX-M-1), CR-Ec and CR gene (blaNDM-1) in aquatic samples from rural and urban settings. Attributes Wastewater Drinking water gene (blaNDM-1) in aquatic samples from rural and urban settings. higher in winter season. 3.4. Seasonal variation in physiochemical properties and prevalence of ESBL-Ec and CR-Ec in the aquatic environments In drinking water samples from urban food mar- kets, the mean concentrations of both total E. coli and ESBL-Ec were the same but in wastewater, the mean concentration of total E. coli was higher than ESBL-Ec. However, the concentration of blaCTX-M-1 and blaNDM-1 in water samples did not vary signiﬁcantly between seasons (Supplementary materials 2). wastewater samples with a mean concentration of 3.48 log10 CFU/100 ml which is higher than the concentrations of CR-Ec in community wastewater reported by other studies in Bangladesh and Thailand (Islam et al., 2017; Thamlikitkul et al., 2019). Previous studies reported that hospital waste is a major source of CR-Ec in the environment, especially where hospital waste is disposed directly to the environment without treatment (King et al., 2020; Lamba et al., 2017; Al Salah et al., 2020; Daoud et al., 2018; Park et al., 2020). However, evidence related to wastewater discharged from urban wet markets as a potential source of CR-Ec has not been widely reported. Therefore, ﬁndings of this study provide important evidence on the contribution of urban food market waste to the environmental spread of AMR. More recently, the WHO has recommended the inclusion of waste- water discharge from urban food markets in LMICs as part of integrated One health surveillance systems for AMR (Matheu et al., 2017). 4. Discussion Though we did not collect any surface water samples adjacent to urban food markets, previous studies reported a high prevalence of ESBL-Ec in the lakes (70% of the isolates and all containing CTX-M genes) and rivers (50% of the isolates and all contain- ing CTX-M-15 genes) around Dhaka city (Haque et al., 2014; Rashid et al., 2015). 8 e context of risk factor analysis for ARB and ARGs we observed use in farm animals on nearby waterbodies has been previously est 0 1 2 3 4 5 6 7 8 9 10 log10 total E.coli count r e t a w e ts a W r e t a w g nik nir D Urban market 0 1 2 3 4 5 6 7 8 9 10 log10 count of ESBL-Ec r e t a w e ts a W r e t a w g nik nir D Urban market 0 1 2 3 4 5 6 7 8 9 10 log10 total E.coli count Drinking water Waste water Pond water River water Rural household Winter Summer 0 1 2 3 4 5 6 7 8 9 10 log10 total E.coli count Drinking water Waste water Pond water River water Poultry farm 0 1 2 3 4 5 6 7 8 9 10 log10 count of ESBL-Ec Drinking water Waste water Pond water River water Rural household 0 1 2 3 4 5 6 7 8 9 10 log10 count of ESBL-Ec Drinking water Waste water Pond water River water Poultry farm Fig. 5. Seasonal distribution of the mean count (log10 CFU/ml) of total E.coli and ESBL-Ec in different aquatic environments. aman et al. Science of the Total Environment 831 (202 Science of the Total Environment 831 (2022) 154890 M. Asaduzzaman et al. CRediT authorship contribution statement Ashbolt, N.J., Amézquita, A., Backhaus, T., Borriello, P., Brandt, K.K., Collignon, P., et al., 2013. Human health risk assessment (HHRA) for environmental development and trans- fer of antibiotic resistance. Environ. Health Perspect. 121 (9), 993–1001. Muhammad Asaduzzaman: Writing original draft, conceptualization of manuscript idea, method development, data collection and analysis, ex- pertise in One Health. Emily Rousham: Conceptualization of the project, funding acquisition, method development, data analysis, supervision of M.A and M.R.I, expertise in global health, review & editing. Leanne Unicomb: Conceptualization of the project, data analysis and discussion, expertise in WASH research, review & editing. Md. Rayhanul Islam: Data analysis and discussion, expertise in statistics, review & editing. Mo- hammed Badrul Amin: Laboratory analysis, expertise in Microbiology, re- view & editing. Mahdia Rahman: Laboratory analysis, expertise in microbiology, review & editing. Muhammed Iqbal Hossain: Laboratory analysis, expertise in microbiology, review & editing. Zahid Hayat Mahmud: Laboratory analysis, expertise in microbiology, review & editing. Mark Szegner: Data analysis and discussion, expertise in GIS map- ping, review & editing. Paul Wood: Conceptualization of the research, method development, data analysis and discussion, expertise in GIS, review & editing. Mohammad Aminul Islam: Conceptualization of the project, funding acquisition, method development, data analysis, supervision of M.A, M.B.A, M.R, M.I.H and M.R.I, expertise in antimicrobial resistance, re- view & editing. Bain, R., Cronk, R., Hossain, R., Bonjour, S., Onda, K., Wright, J., 2014. Global assessment of exposure to faecal contamination through drinking water based on a systematic review. Trop. Med. Int. Health 19 (8), 917–927. Baker-Austin, C., Wright, M.S., Stepanauskas, R., McArthur, J., 2006. Co-selection of antibi- otic and metal resistance. Trends Microbiol. 14 (4), 176–182. Bevan, E.R., Jones, A.M., Hawkey, P.M., 2017. Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype. J. Antimicrob. Chemother. 72 (8), 2145–2155. Bezabih, Y.M., Sabiiti, W., Alamneh, E., Bezabih, A., Peterson, G.M., Bezabhe, W.M., et al., 2021. The global prevalence and trend of human intestinal carriage of ESBL-producing Escherichia coli in the community. J. Antimicrob. Chemother. 76 (1), 22–29. Canales, R.A., Wilson, A.M., Pearce-Walker, J.I., Verhougstraete, M.P., Reynolds, K.A., 2018. Methods for handling left-censored data in quantitative microbial risk assessment. Appl. Environ. Microbiol. 84 (20). Cantón, R., González-Alba, J.M., Galán, J.C., 2012. CTX-M enzymes: origin and diffusion. Front. Microbiol. 3, 110. Connor, R., Renata, A., Ortigara, C., Koncagül, E., Uhlenbrook, S., Lamizana-Diallo, B.M., et al., 2017. Wastewater: the untapped resource. Declaration of competing interest The authors declare that they have no known competing ﬁnancial inter- ests or personal relationships that could have appeared to inﬂuence the work reported in this paper. Role of the funding source The funding agency had no role in study design; in the collection, anal- ysis and interpretation of data; in the writing of the report; and in the deci- sion to submit the article for publication. 5. Conclusion The study was supported by the Antimicrobial Resistance Cross Council Initiative supported by the seven research councils in partnership with the Department of Health and Department for Environment, Food and Rural Affairs (grant NE/N019555/1). MA was supported by National Institutes of Health Fogarty International Center Global Health Equity Scholars program, grant number: D43TW010540 and University of Oslo, Norway. The International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) is thankful to the governments of Bangladesh, Canada, Sweden, and the UK for providing core/unrestricted support. AMR is a major threat to planetary health, which requires an ecosys- tem approach and demands One Health solutions. Our study ﬁndings provide an insight into the waterborne transmission of AMR by mapping and analyzing the spatiotemporal distribution of ARB and ARGs in dif- ferent aquatic environments in Bangladesh. Discharge of untreated wastewater from rural households, poultry farms and urban markets to the open environment is a signiﬁcant contributor to pollution of sur- face water bodies with antibiotic-resistant bacteria and genes. Future interventions are needed to reduce the burden of AMR in the environ- ment. Such interventions could include improved biosecurity practices, particularly waste disposal systems, along with improved water, sanita- tion and hygiene infrastructure in rural and in urban areas, as well as improved practices for intensive poultry farming. GIS based mapping of AMR using quantitative microbiological data from connected environmental compartments can provide valuable insights into the dy- namics of AMR in an ecological setting and this could be an important step in addressing environmental dimensions of AMR through tailored mitigation measures. References Ahammad, Z.S., Sreekrishnan, T., Hands, C., Knapp, C., Graham, D., 2014. Increased water- borne blaNDM-1 resistance gene abundances associated with seasonal human pilgrim- ages to the upper Ganges River. Environ. Sci. Technol. 48 (5), 3014–3020. Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2022.154890. Al Salah, D.M.M., Ngweme, G.N., Lafﬁte, A., Otamonga, J.P., Mulaji, C., Poté, J., 2020. Hos- pital wastewaters: a reservoir and source of clinically relevant bacteria and antibiotic re- sistant genes dissemination in urban river under tropical conditions. Ecotoxicol. Environ. Saf. 200, 110767. Ethical statement In our study, seasonal variation was only evident in the case of CR-Ec in river water which had a higher prevalence and concentration in sum- mer compared to winter. We did not ﬁnd seasonal variation in the prev- alence and concentration of ESBL-Ec or CR-Ec in any other water systems in the urban or rural settings, though CR-Ec prevalence was low. We also investigated a small number of physicochemical parame- ters such as temperature, pH and electrical conductivity of selected water bodies to explore potential associations with ARB concentration across seasons. The increase in both pH and electrical conductivity in winter indicates a higher concentration of metals, minerals and other dissolved solutes that may be derived from waste (Kumar and Prabhahar, 2012; Rashid and Romshoo, 2013). We observed a signiﬁ- cant increase in surface water temperature in the summer compared to winter, which might be a potential effect modiﬁer to the changes in con- ductivity, along with increased rainfall. The widespread prevalence of antibiotic resistant organisms across aquatic environments due to continuous shedding of antibiotic residues from nearby hospitals, unregulated farm practices along with repeated human anthropogenic activities could be the possible reasons for not observing any seasonal effects on prevalence and abundance of AMR in aquatic environments. Ethical approval for the study was obtained from the Institutional Re- view Board of icddr,b, Bangladesh (PR-16071) and the ethics committee of Loughborough University, UK (R17-P037). Written informed consent was given by participants to collect water, wastewater and surface water samples on their premises after receiving written and verbal information in Bangla about the study. Participants were informed of their right to with- draw from the study. Community stakeholders (the local government veter- inary ofﬁce and government community centres in Mirzapur) were also informed about the study as were market authorities. 4. Discussion 0 1 2 3 4 5 6 7 8 9 10 log10 count of ESBL-Ec Drinking water Waste water Pond water River water Rural household 0 1 2 3 4 5 6 7 8 9 10 log10 total E.coli count Drinking water Waste water Pond water River water Rural household Winter Summer 0 1 2 3 4 5 6 7 8 9 10 log10 total E.coli count Drinking water Waste water Pond water River water Poultry farm 0 1 2 3 4 5 6 7 8 9 10 log10 count of ESBL-Ec Drinking water Waste water Pond water River water Poultry farm 0 1 2 3 4 5 6 7 8 9 10 log10 count of ESBL-Ec r e t a w e ts a W r e t a w g nik nir D Urban market Fig. 5. Seasonal distribution of the mean count (log10 CFU/ml) of total E.coli and ESBL-Ec in different aquatic environments. use in farm animals on nearby waterbodies has been previously established (Landers et al., 2012). In the context of risk factor analysis for ARB and ARGs, we observed multiple practices that are likely to contribute to contamination of the aquatic environment, mostly related to biosecurity and waste disposal such as the disposal of wastewater from farms into adjacent ponds and di- rect disposal of household liquid waste into the nearby ditches. We also ob- served high antibiotic use in rural poultry farms and abundance of ESBL-Ec in the adjacent wastewater samples. Nevertheless, these associations were not signiﬁcant due to small sample size although the effect of antibiotic One limitation of our study is that sampling was conﬁned to one urban and one rural locality. 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https://openalex.org/W3182984525	https://zenodo.org/records/7995780/files/Orientation-related%20twinning%20and%20dislocation%20glide%20in%20a%20cantor%20high%20entropy%20alloy%20at%20room%20and%20cryogenic%20temperature%20studied%20by%20in%20situ%20TEM%20straining.pdf	English	null	Orientation-related twinning and dislocation glide in a cantor high entropy alloy at room and cryogenic temperature studied by in situ TEM straining	Materials chemistry and physics	2,021	cc-by	8,634	H I G H L I G H T S • In situ TEM straining experiments performed on a Cantor alloy (HEA) at room and cryogenic temperature. • Plastic mechanisms are observed in real time at the onset of plasticity. • In situ TEM straining experiments performed on a Cantor alloy (HEA) at room and cryogenic temperature. • Plastic mechanisms are observed in real time at the onset of plasticity. • Twinning is more frequently observed at liquid nitrogen temperature. p y • Twinning is more frequently observed at liquid nitrogen temperature. p y • Twinning is more frequently observed at liquid nitrogen temperature. • The activation of twinning or perfect dislocation glide mainly depends on crystal orientation. • Both modes of plasticity can therefore occur jointly in random oriented polycrystals, even at early stages of deformation. • The activation of twinning or perfect dislocation glide mainly depends on crystal orientation. • Both modes of plasticity can therefore occur jointly in random oriented polycrystals, even at early stages of deformati Daniela Oliveros a,b, Anna Fraczkiewicz c, Antonin Dlouhy d, Chen Zhang e, Hengxu Song e, Stefan Sandfeld e, Marc Legros a,* Daniela Oliveros a,b, Anna Fraczkiewicz c, Antonin Dlouhy d, Chen Zhang e, Hengxu Song e, Stefan Sandfeld e, Marc Legros a,* a CEMES-CNRS, 29 Rue J. Marvig, 31055, Toulouse, France b Universit´e de Toulouse, 118 Route de Narbonne, 31400, Toulouse, France c MINES Saint-Etienne, Universit´e de Lyon, CNRS, UMR 5307 LGF, Centre SMS, 42023, Saint-Etienne, France d Institute of Physics of Materials and CEITEC IPM, Czech Academy of Sciences, 616 62, Brno, Czech Republic e Forschungszentrum Jülich GmbH, Institute for Advanced Simulations: Materials Data Science and Informatics (IAS-9), 52425, Jülich, Germany a CEMES-CNRS, 29 Rue J. Marvig, 31055, Toulouse, France * Corresponding author. E-mail address: marc.legros@cemes.fr (M. Legros). A R T I C L E I N F O Keywords: In situ TEM High entropy alloy Plasticity Twinning Dislocation In situ straining experiments were performed in a TEM on an equimolar CoCrFeMnNi (Cantor) high entropy alloy at room and cryogenic temperature. Perfect and partial dislocation activity were recorded in both cases. Twinning directly follows the development of partial dislocation shearing that has various origins (perfect dislocation splitting, anchoring). It is shown that, although twinning is more frequently observed at liquid ni trogen temperature, its prevalence depends mainly on crystal orientation. As a result, twinning and perfect dislocation plasticity are likely to occur jointly in random oriented polycrystals, even at early stages of deformation. HEA to call this new class of metallic alloys. Materials Chemistry and Physics 272 (2021) 124955 Materials Chemistry and Physics 272 (2021) 124955 * Corresponding author. E-mail address: marc.leg Orientation-related twinning and dislocation glide in a cantor high entropy alloy at room and cryogenic temperature studied by in situ TEM straining Orientation-related twinning and dislocation glide in a cantor high entropy alloy at room and cryogenic temperature studied by in situ TEM straining 1. Introduction On the contrary, in low-SFE metals such as Cu, Ag [16] the large splitting of perfect dislocations into partials favors planar glide, extended stacking fault development and twinning. This can be beneficial as it offers both additional deformation modes and hardening possibilities, but may also lead to structural instability via martensitic transformation as in low-SFE steels [17]. Rectangular (3 × 1.5 × 0.5 mm) samples were cut from these ingots and slabs using electro-discharge machining. They were mechanically thinned down to about 30 μm with SiC papers. Final electropolishing was made in a Struers Tenupol twin-jet polishing unit using a 10% Perchloric acid-90% Ethanol electrolyte to create thin regions around a central hole (Fig. 1a). X-ray Energy Dispersive Spectroscopy (EDS) was performed on the thicker part of the polished zone, and within the ac curacy of the technique (1–2% without the use of standards) the equi molar composition of samples was verified. i These rectangular samples are then glued with cyanoacrylate onto Cu grids specially designed for Gatan straining holders (Fig. 1c). In this study, a cryo-straining holder served both for liquid nitrogen (LN2) and room temperature (RT) in situ experiments. The so-called “LN2 experi ments” were actually performed at 100–105 K, as the sample is cooled down through a cold finger running through the TEM holder and con nected to its LN2 reservoir. 100 K is close to the minimum reachable with this holder, with some fluctuations depending on the outside tempera ture and the level of vacuum reached in the reservoir shell. We used it without temperature regulation, at the minimum attainable temperature for a given experiment. More details about sample preparation and in situ straining experiments can be found in videos posted online [22,23]. y In HEAs, the situation is more complex. The fact that the position of the various chemical species is random (including in the slip plane) precludes any easy prediction of the SFE. Recent Transmission Electron Microscopy (TEM) observations of dissociated dislocations lead to SFE estimations in the range of 18–27 mJ m−2 [18], but larger fluctuations have been observed and may extend this range [19]. These values come from static observations. Overall, the proportion of twinning versus perfect dislocation glide remains a question as both systems have been observed across a wide temperature range, and the mechanisms by which these two kinds of plasticity unfold in fcc HEAs are still ques tioned. 1. Introduction Since the early 2000s, a considerable body of literature has emerged to explore their processing and properties. In most recent review arti cles, e.g. Ref. [5], many poorly understood properties of these alloys are exposed and pave the way for future studies. As for other HEAs or MEAs (Medium Entropy Alloys containing 3 or 4 principal elements) of fcc structure, the mechanical properties of the CoCrFeMnNi (Cantor) alloy and its family are remarkable [6]. In particular, their toughness remains very high at cryogenic temperatures [7]. The principal hypothesis sug gested for this intriguing property in literature [7–11] is the propensity of fcc-structured HEAs to twin at low temperatures and up to 500 ◦C [8]. However, the few in-depth studies on the subject give very partial High Entropy Alloys (HEA) were born from the idea that regular alloys constituted of one or two main elements represented a very small fraction of the combinatorial possibilities offered by the many metallic elements of the periodic table [1,2]. i Their name was derived from the early belief that the configurational entropy of mixing many different elements would overcome the enthalpy gain of stabilizing intermetallic phases. This concept has been challenged since then [3,4], and although other names, may be more relevant, have been suggested (MPEAs- for Multiple Principal Element Alloys), many authors still rely on the term Materials Chemistry and Physics 272 (2021) 124955 D. Oliveros et al. answers as only a few testing conditions were reported, even if a potentially large number of influential parameters (temperature, orientation, strain, stacking fault energy …) exist [9–13]. microstructure, with large grains (≈50 μm). The lowest dislocation density was observed in the fully recrystallized alloy (IPM, Brno), although no systematic measurements were performed due to grain-to- grain variations. In fact, the mechanical properties of fcc metals rely heavily on the behavior of the main dislocation slip systems that, in a vast majority of deformation conditions, consist of a/2<110> type dislocations gliding on (111)-type planes. These dislocations are asymmetrically split into two a/6<112> dislocations separated by a stacking fault ribbon, the width of which is inversely proportional to the stacking fault energy (SFE) [14]. In high-SFE metals such as Al, the separation of both partial is very small, favoring the cross-slip of perfect dislocations and pre venting twinning in most of the cases [15]. 1. Introduction The purpose of this study is an in-depth characterization of the mechanisms of plastic deformation, on the basis of an extensive in situ TEM study, with an analysis of more that 60 different grain orientations. The study was performed on an equimolar CoCrFeMnNi fcc HEA (Cantor alloy) both at room (RT) and liquid nitrogen (LN2) temperatures. We show that low temperature favors twinning but that grain orientation is critical to promote this type of plasticity. Finite Element Method (FEM) calculations made with CATIA® illustrate the fact that the thin regions along the straining axis are those where the stress reaches a maximum (Fig. 1b). The FEM calculation is using a linear elastic material model. Therefore, all forces and stresses are linearly related and can therefore be easily scaled to larger elastic stresses. Moreover, the resolved shear stress (RSS) τ acting on dislocations moving during in situ tests was also estimated as the line tension stress: τ = μb/R, where b is the Burgers vector and R the radius of curvature of the dislocation. The shear modulus μ of the Cantor alloy at RT and Moreover, the resolved shear stress (RSS) τ acting on dislocations moving during in situ tests was also estimated as the line tension stress: τ = μb/R, where b is the Burgers vector and R the radius of curvature of the dislocation. The shear modulus μ of the Cantor alloy at RT and LN2 temperature was taken from the work by Haglund et al. [24]. Fig. 1. Typical shape and stress concentra tion around in the electron-transparent zone in an electropolished sample for in situ TEM straining. a) Low magnification TEM image, b) FEM stress concentration in the thin re gions around the hole (FEM calculation made with CATIA V5.6, applied force of 90 N, Min (blue)/Max (red) Von Mises Stress = 1.5/65 kPa), c) sample glued on a straining Cu grid. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Fig. 1. Typical shape and stress concentra tion around in the electron-transparent zone in an electropolished sample for in situ TEM straining. a) Low magnification TEM image, b) FEM stress concentration in the thin re gions around the hole (FEM calculation made with CATIA V5.6, applied force of 90 N, Min (blue)/Max (red) Von Mises Stress = 1.5/65 kPa), c) sample glued on a straining Cu grid. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2. Material and methods p y g Under in situ deformation, orientation of the active grain was determined by capturing 3 to 4 two-beam diffraction patterns (DPs) using the single tilt function of the TEM (a JEOL 2010 LaB6 operating at 200 kV). DPs, micrographs and videos were all recorded using a Megaview III camera from Soft Imaging System (now EMSIS) and stored on hard drives. Unless stated otherwise, the straining axis is vertical in all the micrographs and videos (Fig. 2a), corresponding to the direction going from pole to pole on the stereographic projection (Fig. 2b) ob tained from the captured diffraction patterns (insert in Fig. 2a). This combination allows us to directly index the activated slip planes (Fig. 2a, c) and, subsequently, the local thickness of the TEM foil (Fig. 2d). PycoTEM, an open source software developed at CEMES, was also used to construct these stereographic projections [25]. During in situ experi ments, short strain pulses (on the order of 10−3 s−1) are applied, sepa rated by longer periods where the dislocation movements are observed. In average, the strain rate of the in situ experiments are in the order of Ingots of CoCrFeMnNi equimolar alloy (Cantor alloy) were prepared from pure metals pellets or powders (purity always higher than 99,9% purity) following two methods. After cold crucible melting and ho mogenization (MINES Saint-Etienne), the ingots were hot forged and annealed for 2 h at 1000 ◦C under vacuum [20]. The second batch of samples were manufactured by arc melting (8.4 × 10−4 Pa Ar atmo sphere) and drop casting at ORNL and provided by the Institute of Physics of Materials in Brno. The Mn weight loss during arc melting is compensated by adding 1 g (for a 475 g ingot) and a Zr pellet was melted before to clean the chamber from residual oxygen. Arc-melted buttons were flipped and re-melted 5 times for homogenization, drop cast in rectangular Cu molds, and solution-annealed 48 h at 1200 ◦C. After being cold-rolled from 12.7 mm down to 4 mm thick slabs, they were annealed 1H at 900 ◦C. For processing details see Ref. [21]. Both processes resulted in a homogeneous single phase Fig. 1. Typical shape and stress concentra tion around in the electron-transparent zone in an electropolished sample for in situ TEM straining. 2. Material and methods a) Low magnification TEM image, b) FEM stress concentration in the thin re gions around the hole (FEM calculation made with CATIA V5.6, applied force of 90 N, Min (blue)/Max (red) Von Mises Stress = 1.5/65 kPa), c) sample glued on a straining Cu grid. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2 Fig. 1. Typical shape and stress concentra tion around in the electron-transparent zone in an electropolished sample for in situ TEM straining. a) Low magnification TEM image, b) FEM stress concentration in the thin re gions around the hole (FEM calculation made with CATIA V5.6, applied force of 90 N, Min (blue)/Max (red) Von Mises Stress = 1.5/65 kPa), c) sample glued on a straining Cu grid. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 2 Materials Chemistry and Physics 272 (2021) 124955 Fig. 2. Orientation and foil thickness measurement during an in situ TEM straining test. a) Dislocation pile up in a grain and corresponding Diffraction Pattern (DP) on the lower right. b) Corresponding stereographic projection (111)-type planes are in red), the activated plane is (-111) in this case. Straining axis is vertical in a) and goes through both north and south poles of the stereographic projection of b). c) Sketch of the dislocation pile up in its (-111) glide plane. d) projected image and measured film thickness: t = Lproj.tan (90-α). In this relation L_proj is the same as in Fig. 2c only for small sample tilt angles which is true for our setup. In this example, Lproj = 430 nm, and = 25◦, so t = 200 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) D. Oliveros et al. Materials Chemistry and Physics 272 (2021) 124955 D. Oliveros et al. Fig. 2. Orientation and foil thickness measurement during an in situ TEM straining test. a) Dislocation pile up in a grain and corresponding Diffraction Pattern (DP) on the lower right. b) Corresponding stereographic projection (111)-type planes are in red), the activated plane is (-111) in this case. Straining axis is vertical in a) and goes through both north and south poles of the stereographic projection of b). 3. Results About 25 in situ TEM straining experiments were carried out at room and LN2 temperatures, representing more than 60 different grain ori entations. Strain increments are imposed on the sample through the motor of the straining holder and observations are made during subse quent relaxation time. Dislocation activity initiates in stressed regions of the foil (Fig. 1c) and from all the usual stress-concentration sites: grain boundaries (GBs), entanglements of sessile dislocations, thin foil edges. This generates localized plasticity that subsequently unravels in the sample. 2. Material and methods c) Sketch of the dislocation pile up in its (-111) glide plane. d) projected image and measured film thickness: t = Lproj.tan (90-α). In this relation L_proj is the same as in Fig. 2c only for small sample tilt angles which is true for our setup. In this example, Lproj = 430 nm, and = 25◦, so t = 200 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 10−4 s−1 to 10−5 s−1. 10−4 s−1 to 10−5 s−1. This leads to an intra-granular, distributed plasticity. When dislocations can easily cross a GB, plastic deformation may remain spread, but this happens rarely. In any opposite case, activation of dislocation sources (in thin foils, only single spiral sources are observed) generally lead to long and dense pile-ups of dislocations (as visible in Figs. 3 and 6) that concentrate the deformation along a restricted set of (111)-type planes. It can be seen that the pile-up in Fig. 3 densifies after an increase in strain, and that the dislocations’ radius of curvature diminishes (e.g. stress increases), even for those dislocations that are situated at the end of the pile-up. Curvature measurements made on the last moving dislocation in this pile-up leads to a Resolved Shear Stress (RSS) of 53 MPa, corresponding to an applied stress of 112 MPa. Such local stress measurement underestimates the overall applied stress as it also in corporates the back stress of the pile-up and a possible friction stress. In Fig. 3b, a dislocation followed by a stacking fault (SF) is emerging from the back of the pile-up. This partial dislocation is followed by others coming from the thinner part of the foil (right side in Fig. 3), with the same Burgers vector. In Fig. 3c, the characteristic grey contrast (with fringes) in regions 1, 2, 3 attests that the initial perfect dislocation pile- Fig. 3. Development of plastic deformation during an in situ TEM experiment performed at 100 K. a) Perfect dislocation with b = a/2 [0–11] and gliding on the (-111) plane blocked by a GB. b) After a strain/stress increment, dislocations move towards the left where they are blocked by a grain boundary (GB). A partial dislocation, leaving a stacking fault (SF) is incorporated in the pile-up. c) Upon unloading, and in a thinner part of the sample, the back of the pile-up is constituted by partial dislocations (b = a/6 [-1-12]) forming a micro-twin. Each image is taken ≈30 min after the preceding one. 3.1. Low temperature plasticity: LN2 temperature experiments 3c, indicated by “R”, where perfect dislocations (coming from the left) dissociate at the interaction with a sessile dislocation that serves as obstacle, and form micro twins (right) is commonly observed. The development of plasticity at LN2 temperature often involves a mix of perfect dislocations and twinning. Fig. 4 shows more snapshots extracted from an in situ TEM straining experiment carried out at 104 K where two pile-ups of perfect disloca tions located on (-1-11) and (-111) planes are visible. Perfect disloca tions in these pile-ups are a/2 [011] (Schmid Factor = 0.02) and a/2 [-1- 10] (Schmid Factor = 0.46) respectively, which means that the later will be much more favored than the former when applying an external stress (always along the vertical axis on the micrographs). One widely split dislocation separated by a stacking fault is visible in the (-111) plane in Fig. 4a. After applying a 2 μm displacement increment on the holder, plastic deformation intensifies on the (-111) plane mainly by the prop agation of partial dislocations that leave longer stacking faults (Fig. 4). The pile-up on (-1-11), mainly formed by perfect dislocations, does not move because its Schmid factor is too low. On the contrary, perfect dislocations moving on (-111) are more active (large Schmid factor), as for partial dislocations (b = a/6 [211] Schmid Factor = 0.47) that pull stacking faults under stress. As seen in Fig. 4b, the inactive perfect dis locations on (-1-11) serve as obstacles to the slip of a/6 [211] partial dislocations on (-111). The darker contrast visible in the middle of the stacking fault is due to a partial dislocation gliding towards the (-1-11) pile-up close to a previously drawn stacking fault. Once a third partial follows the same path, a micro-twin is created, provided the partial dislocations glide on adjacent planes and have the same Burgers vector. At room temperature, perfect dislocations are however more often observed. Fig. 6 shows a typical set of perfect a/2 <110> dislocations gliding on (1–11) planes. Dense pile-ups develop against strong obsta cles such as GBs. As for low temperature experiments, we also measured dislocation curvatures on the last dislocation of the pile-up and this returned a much lower value of the RSS: 22 MPa in this case, corre sponding to 55 MPa of applied stress. 3.1. Low temperature plasticity: LN2 temperature experiments Fig. 3 illustrates the beginning of an in situ TEM straining experiment at 100 K. First, the applied stress mobilizes dislocations that are left over from a denser microstructure in the annealed alloy (top of Fig. 3b and c). Fig. 3. Development of plastic deformation during an in situ TEM experiment performed at 100 K. a) Perfect dislocation with b = a/2 [0–11] and gliding on the (-111) plane blocked by a GB. b) After a strain/stress increment, dislocations move towards the left where they are blocked by a grain boundary (GB). A partial dislocation, leaving a stacking fault (SF) is incorporated in the pile-up. c) Upon unloading, and in a thinner part of the sample, the back of the pile-up is constituted by partial dislocations (b = a/6 [-1-12]) forming a micro-twin. Each image is taken ≈30 min after the preceding one. Fig. 3. Development of plastic deformation during an in situ TEM experiment performed at 100 K. a) Perfect dislocation with b = a/2 [0–11] and gliding on the (-111) plane blocked by a GB. b) After a strain/stress increment, dislocations move towards the left where they are blocked by a grain boundary (GB). A partial dislocation, leaving a stacking fault (SF) is incorporated in the pile-up. c) Upon unloading, and in a thinner part of the sample, the back of the pile-up is constituted by partial dislocations (b = a/6 [-1-12]) forming a micro-twin. Each image is taken ≈30 min after the preceding one. 3 Materials Chemistry and Physics 272 (2021) 124955 D. Oliveros et al. micro-twinning upon further straining. The transformation of perfect dislocation into micro-twins and inversely has also been observed after reactions on obstacles (R in Fig. 3), usually forest dislocations. Again, we did not observe a clear transition from one deformation mode to the other related to the amount of strain (density of dislocation increasing nor stress concentration due to strain hardening). Twinning can occur right at the onset of plasticity or after a few percent of deformation (typically, in situ straining tests do not extend beyond 10% strain). up is being replaced by a micro-twin (which implies that partial dislo cations lie on adjacent planes rather than on the exact same one). The way this transformation occurred is not yet identified, but the reaction seen in Fig. 3.1. Low temperature plasticity: LN2 temperature experiments Again, this value is underestimated as it does not take into account the back-stress from the first dislocations of the pile-up nor possible friction stress. Nonetheless, it is worth noting that this stress value, measured in similar conditions at low and room temperature, reproduces well the macroscopic observations: in Cantor’s alloy, the stress needed to deform the alloy increases when the tem perature decreases into the cryogenic area. It can also be seen in Fig. 6 that smaller obstacles, such as immobile dislocations are usually overcome by a few (less than ten) dislocations. Other “invisible” obstacles, such as impurities or short-range order chemical variations are also probably responsible for the cusps seen frequently on individual or collective dislocations (pile-ups and shear bands). These cusps impose much lower radii of curvature on dislocation lines (perfect and partial) and are observed much more frequently at low temperature than at room temperature. The strong curvature is an indication of the larger stress needed for the dislocations to overcome these obstacles. A quantitative and statistical study is currently carried out to assess the strength of the various obstacles present in this specific alloy. 3.2. Room temperature plasticity As a result of the development of pile-ups, the deformation is concentrated in several shear bands where one or several (111) planes are heavily sheared. This concentration is partially lowered at GBs where some alternate glide planes are activated, unless a closely orientated slip system is available in the next grain. At room temperature, incipient plasticity can also develop through partial dislocation activity and twinning. Fig. 5 shows several snapshots extracted from an in situ TEM straining experiment carried out at 300 K. Each “dark triangle” in Fig. 5a is a dissociated a/2 [011] dislocation gliding on the (-1-11) plane. As the applied strain (and thus stress) in creases, the dissociation expands too (Fig. 5b and c). The process ap pears reversible as a decrease of the applied stress causes the dissociation to shrink back almost to the initial configuration (Fig. 5d). Continuing the straining experiment spurs the extension of the stacking faults separating both partials to the point where a recombination be comes impossible. Leading and trailing partial dislocations interact with other defects (dislocations on other glide planes, GBs) and their sepa ration distance increases until their elastic interaction is negligible. The network formed by these stacking faults on two different sets of glide planes (Fig. 5e and f) constitutes a large array of potential obstacles for other dislocations, perfect or partial. We have summarized all these observations in Fig. 7. The presented graph identifies the crystallographic orientation of each tested grain in all the strained samples: each dot corresponds to the straining axis in a grain that has been deformed at room or LN2 temperature. The pink (resp. green) areas regroup the grains in which only perfect dislocation glide (resp. only twinning) has been observed. The yellow area regroups the grains in which both modes of deformation co-existed during the straining experiment. It can be seen that both deformation modes are active at both temperatures, but that twinning is favored at low tem perature. To be more precise, very few grains have demonstrated twinning-only deformation (green areas), but grains where both twin ning and perfect dislocation activity co-exist (yellow area) represent a These long stacking faults ribbons and individual partials lead to 4 Fig. 4. Mix of perfect and partial dislocation development of plastic deformation during a TEM experiment performed at 104 K in a pa foil where the thickness is 165 nm. Fig. 4. Mix of perfect and partial dislocations in the development of plastic deformation during an in situ TEM experiment performed at 104 K in a part of the foil where the thickness is 165 nm. a) Low magni fication of a zone deforming plastically., containing perfect dislocations pile-ups (P) and one widely split dislocation separated by a stacking fault (SF). b) Upon straining, glide of partial dislocations on (-111) leaves longer stacking faults. Note that dislocation lines are in general more cusped than at room temperature. Fig. 4. Mix of perfect and partial dislocations in the development of plastic deformation during an in situ TEM experiment performed at 104 K in a part of the foil where the thickness is 165 nm. a) Low magni fication of a zone deforming plastically., containing perfect dislocations pile-ups (P) and one widely split dislocation separated by a stacking fault (SF). b) Upon straining, glide of partial dislocations on (-111) leaves longer stacking faults. Note that dislocation lines are in general more cusped than at room temperature. 3.2. Room temperature plasticity In other words, for randomly orientated grains, observing perfect dislocations will be easier at room temperature. larger fraction of the orientations at low temperature (Fig. 7b). Inversely, at room temperature (Fig. 7a), a larger zone of the standard triangle is occupied by the pink cloud, corresponding to straining axis orientations leading to perfect dislocation deformation only. The same pink region is smaller at low temperature. In other words, for randomly orientated grains, observing perfect dislocations will be easier at room temperature. to explain the capacity of a metal or an alloy to deform by twinning is the stacking fault energy (SFE). For instance, aluminum, characterized by a very high SFE (over 100 mJ/m2 [26–28]) does not twin unless very specific circumstances [15]. On the other hand, a low SFE favors twin ning, such as observed in copper, brass, …[29,30], but also in low SFE austenite in austenitic stainless steels from Fe–Cr–Ni system [31,32]. Zaddach et al. [18], using ab-initio calculations, estimated the stacking fault energy for the equimolar CoCrFeMnNi alloy between 18 and 27 mJ m−2. These authors have also shown a strong variation of SFE upon deviation of chemical composition from the equimolar one [18]. Experimentally, average values were given around 30 mJ m−2 [33], but very large fluctuations of dissociated dislocations suggest that either this SFE value varies along a given dislocation line or that a “local SFE” may have to be considered [19]. Fluctuations of SFE may arise from local variation of chemical composition that could itself be influenced by the 3.2. Room temperature plasticity a) Low fication of a zone deforming plastically., con perfect dislocations pile-ups (P) and one split dislocation separated by a stacking fau b) Upon straining, glide of partial dislocat (-111) leaves longer stacking faults. No dislocation lines are in general more cusped room temperature. Fig. 4. Mix of perfect and partial dislocations in the development of plastic deformation during an in situ TEM experiment performed at 104 K in a part of the foil where the thickness is 165 nm. a) Low magni fication of a zone deforming plastically., containing perfect dislocations pile-ups (P) and one widely split dislocation separated by a stacking fault (SF). b) Upon straining, glide of partial dislocations on (-111) leaves longer stacking faults. Note that dislocation lines are in general more cusped than at room temperature. D. Oliveros et al. Materials Chemistry and Physics 272 (2021) 124955 Materials Chemistry and Physics 272 (2021) 124955 g. 5. Splitting of perfect dislocation leading to extended stacking faults during a 300 K in situ TEM experiment. a)-c) Swelling dissociation under increasing strain aken at t = 0, 18′ and 20′ respectively). d) Shrinking dissociation under decreasing strain (t = 23′). t1 shows the same trailing partial dislocation. e) Development of tended stacking faults, easily recognizable by their dark grey fringe contrast, on (-1-11) and (111) planes during the experiment. f) After the experiment and the lease of the strain, most of the extended stacking faults shrunk and disappeared. A few perfect dislocations are also visible (P). Oliveros et al. Fig. 5. Splitting of perfect dislocation leading to extended stacking faults during a 300 K in situ TEM experiment. a)-c) Swelling dissociation under increasing strain (taken at t = 0, 18′ and 20′ respectively). d) Shrinking dissociation under decreasing strain (t = 23′). t1 shows the same trailing partial dislocation. e) Development of extended stacking faults, easily recognizable by their dark grey fringe contrast, on (-1-11) and (111) planes during the experiment. f) After the experiment and the release of the strain, most of the extended stacking faults shrunk and disappeared. A few perfect dislocations are also visible (P). larger fraction of the orientations at low temperature (Fig. 7b). Inversely, at room temperature (Fig. 7a), a larger zone of the standard triangle is occupied by the pink cloud, corresponding to straining axis orientations leading to perfect dislocation deformation only. The same pink region is smaller at low temperature. 4. Discussion The present study underlines the fact that two modes of plasticity, mechanical twinning (including glide of isolated partial dislocations) and perfect dislocation glide, coexist in the equimolar CoCrFeMnNi alloy at both tested temperatures: around 100 K (LN2, liquid nitrogen) and 300 K (RT, room temperature). The key parameter traditionally invoked 5 Materials Chemistry and Physics 272 (2021) 124955 D. Oliveros et al. D. Oliveros et al. expected that stacking fault extension and then twinning would occur mainly in the red zone, near 001. This is roughly verified, with twinning only occurring near the 001 pole at both temperatures and perfect dislocation glide taking place mainly near the 111 axis. What is sur prising here is the extent of mixed modes (twin + perfect dislocations) towards this 111 pole, especially at low temperatures. Fig. 6. Pile-ups of perfect dislocations during a 300 K in situ TEM straining experiment. Both pile-ups are formed on two parallel (1–11) planes against a GB located further right and up in the sample (not visible in the picture) (b = a/ 2 [011], (1–11) glide plane). Note that dislocation lines are more rounded (less pinning points) than at low temperature. A possible explanation for this extension of mixed modes lies in the local deviations to the macroscopic straining axis that occur in our thin foil configuration where a hole is punched in the center (Fig. 1). The straining axis has a tendency to align parallelly to the edges of the hole in the thinnest areas. Therefore, in a given experiment where several grains are stressed at once, we collect grain orientations where the actual tensile axis is within ±15◦of the macroscopic one. As a consequence, reporting the macroscopic tensile axis in a given grain introduces an uncertainty regarding the actual Escaig stress in this grain where one mode should prevail over the other. This explains why the yellow zone in Fig. 7 (corresponding to an expanded region around the “neutral” white zone in Fig. 8) is much wider. As a consequence, the single mode zones (green and pink in Fig. 7) are reduced to areas very close to the 111 and 001 orientations. Fig. 6. Pile-ups of perfect dislocations during a 300 K in situ TEM straining experiment. 4. Discussion Both pile-ups are formed on two parallel (1–11) planes against a GB located further right and up in the sample (not visible in the picture) (b = a/ 2 [011], (1–11) glide plane). Note that dislocation lines are more rounded (less pinning points) than at low temperature. The splitting of partial dislocations that takes place in Fig. 5 is emblematic of the mechanism predicted in Fig. 8. This grain however, labelled #16 in Fig. 7a, is not located in a splitting-favored zone of Fig. 8 (although close to the white separation line). Several reasons may explain this apparent discrepancy. As explained above, the straining axis may not be exactly vertical in this particular grain. The neighboring grains may also impose a deviation of the applied straining axis. The main reason is probably that the (-1-11) plane in which this dissociation occurs is not the one most favored by the Schmid factor. As Fig. 8 only addresses the behavior of the preferred glide system (highest Schmid factor) we should re-draw the shear stress directions for this specific glide system, which is beyond the scope of this paper [35]. presence of dislocations, although this has not been clearly evidenced yet, even from recent in situ straining experiments at room temperature [34]. From our experiments, it is clear that an applied shear stress is able to split the asymmetric dissociation of perfect dislocations (Fig. 8), even at or just before the onset of plastic deformation, as exemplified in Fig. 5. i This can be rationalized in terms of projected local stress state onto activated dislocations. Fig. 8 reproduces the variation of the Peach- Kohler shear stress acting on both partial dislocations bp1 and bp2 that compose a perfect dislocation of Burgers vector b gliding on a (xy) plane. For a tensile test, the stress difference τ′d between the shear acting on the edge component of each partial dislocations (σ1 yz and σ2 yz.) will favor the constriction of dislocation b when positive or its dissociation when negative. This so-called Escaig stress is then plotted in a standard ste reographic triangle for the most favorable slip system (highest Schmid factor). τ′d reaches the strongest negative values (red zone) near the 001 orientation. In the blue region, τ′d is positive and the applied stress tends to reduce the splitting [35]. 5. Conclusions In situ TEM straining experiments were carried out at room and near liquid nitrogen temperature on an equimolar CoCrFeMnNi (“Cantor alloy”) austenitic high entropy alloy. It was found that: • Twinning and perfect dislocation glide occurred at both tempera tures, and could be triggered at the onset of plasticity; • The grain orientation with respect to the loading axis is the primary factor influencing the mode of deformation; A decrease of SFE at low temperature would favor this effect, as anticipated by some authors from their ab initio calculations [39]. However, as seen before, not only simulations of this very complex class of alloys are to be validated by experiments, but those have revealed that variations of the SFE, because of local order or segregation effects can impact the dissociation by a factor 2 or more. l • In particular, the dissociation of perfect dislocation seems a common mechanism leading to the development of extended stacking faults. These extended stacking faults serve as seeds to the expansion of mechanical twins; • The more frequent occurrence of twinning at low temperature could be the result of a lowering of the stacking fault energy and a more effective pinning of trailing partial dislocations. In all the straining experiments, we observed that the pinning points arising from local chemical or order variations (different from forest/ dislocation type) are stronger at low temperature. This feature is clearly evidenced by the fact that they impose steeper curvatures on mobile dislocations. As stated before, the motion of dislocation requires larger stresses at 100 K (112 MPa vs 53 MPa, as measured on tail dislocations in moving pile-ups). Because these pinning points act similarly on partial or perfect dislocation, this action, combined with a low SFE will equally pin the leading and trailing partials of dissociated dislocations. We previously saw that a random orientation of the applied shear will favor the motion of the two partial dislocations of a dissociated perfect dislocation in the same direction (blue region in Fig. 8). This combined action of the stress on a pair of dislocations favors the overcoming of an obstacle. In the case where the stress acts to separate the dislocations, the stacking fault will pull the two partial dislocations together. In this context weak obstacles will not separate these partial dislocations. 4. Discussion The fact that low Schmid factor slip systems are activated is not uncommon as the alloy, even in the annealed state, contains many remaining dislocations. These remnant dislocations can be activated even if their Schmid factor is not maximum, which seems to be the case here as their velocity under stress is much lower than in other grains. Because of all these factors, concomitantly observing two modes of deformation (twinning and perfect dislocation glide) is not surprising. What our experiments show is that activation of a given system is largely influenced by the grain orientation with respect to the local straining axis, but that local factors such as the initial microstructure, may blur this picture and mix deformation modes in many cases. We also show that twinning is slightly favored at low temperature, but this is a second The fact that low Schmid factor slip systems are activated is not uncommon as the alloy, even in the annealed state, contains many remaining dislocations. These remnant dislocations can be activated even if their Schmid factor is not maximum, which seems to be the case here as their velocity under stress is much lower than in other grains. Because of all these factors, concomitantly observing two modes of deformation (twinning and perfect dislocation glide) is not surprising. What our experiments show is that activation of a given system is largely influenced by the grain orientation with respect to the local straining axis, but that local factors such as the initial microstructure, may blur this picture and mix deformation modes in many cases. We also show that twinning is slightly favored at low temperature, but this is a second Comparing this standard triangle to those of Fig. 7, it could be 6 Fig. 7. Orientation dependence of micro-twinning vs perfect dislocation glide in standard triangles at room (a) and LN2 (b) temperatures. Fig. 7. Orientation dependence of micro-twinning vs perfect dislocation glide in standard triangles at room (a) and LN2 on dependence of micro-twinning vs perfect dislocation glide in standard triangles at room (a) and LN2 (b) temperatures. 6 Fig. 7. Orientation dependence of micro-twinning vs perfect dislocation glide in standard triangles at room (a) and LN2 (b) temperatures. D. Oliveros et al. Materials Chemistry and Physics 272 (2021) 124955 Fig. 8. Escaig split of two partial dislocations in function of the straining direction in tension. order effect. long stacking faults (as in Fig. 4) and twins in the crystal as the defor mation proceeds [6]. As demonstrated in our experiments, the presence of extended stacking faults favors the subsequent development of me chanical twinning. In contrast to the previous studies, we did not observe cases where twinning would be triggered by cross-slip [13], nor that a certain threshold of stress is required to activate this deformation mode [36]. In fact, the stress levels measured from the curvature radii of the mobile dislocations were very low, which is not surprising at the onset of plastic deformation. Even if these curvatures were measured in the tail of pile-ups where the back-stress of front dislocations reduces the local resolved shear stress, we are very far from the hundreds of MPa that are supposed to be needed to trigger twinning. Twinning is activated not only in zones where stacking faults were observed at the beginning of the deformation, but also after the reaction of perfect dislocations on forest-type obstacles (Fig. 3c). This observa tion reinforces the former hypothesis that twinning may develop once a sufficient density of obstacles exist (strain hardening, leading to a suf ficient amount of stress [36]) or if the existing obstacles are oriented conveniently to create the right reaction with incoming dislocations (through cross-slip for instance Ref. [13]). It can also be argued that working in a thin foil favors the splitting of dislocations as image forces act in opposite direction on both sides of the crystal [37]; thus, nucleation of partial dislocations can also be favored compared to perfect ones in thin foils [38]. Yet, both effects will influ ence the behavior of dislocations only in very thin foils (30–50 nm and below) which is far from our experimental conditions: most of our an alyses were carried out in 100–300 nm thick regions. Also, as all experimental conditions were the same or very similar between all the samples that underwent in situ straining experiments (straining rate - typically in the range of 10−5 s−1, mostly in relaxation mode, sample preparation …), the increased proportion of twins in samples tested at low temperature is indicative of a real trend. 4. Discussion Left: bp1 and bp2 (a/6 [112] type) compose a perfect dislocation b (a/2 [110] type) split in the (xy) plane. τ′d is the difference between the Peach Kohler stresses acting on the edge components of bp1 and bp2. Right: sign and amplitude of τ′d in function of the direction of the applied stress in the standard stereographic triangle. Fig. 8. Escaig split of two partial dislocations in function of the straining direction in tension. Left: bp1 and bp2 (a/6 [112] type) compose a perfect dislocation b (a/2 [110] type) split in the (xy) plane. τ′d is the difference between the Peach Kohler stresses acting on the edge components of bp1 and bp2. Right: sign and amplitude of τ′d in function of the direction of the applied stress in the standard stereographic triangle. 5. Conclusions However, if obstacles become stronger, as it seems to be the case at low temperature, the probability that the trailing partial is retained by one of them increases. 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https://openalex.org/W2953741475	https://www.ped-perinatology.ru/jour/article/download/902/794	Russian	null	Improvement of therapy efficacy in children with atopic dermatitis due to emollient complex	Rossijskij vestnik perinatologii i pediatrii	2,019	cc-by	5,993	Активные исследования атопического дерматита выявляют новые звенья патогенеза и подтверждают необходимость комплексного подхода к лечению, включающего различные формы выпуска увлажняющих препаратов (крем и средство для купания). Активные исследования атопического дерматита выявляют новые звенья патогенеза и подтверждают необходимость комплексного подхода к лечению, включающего различные формы выпуска увлажняющих препаратов (крем и средство для купания) Цель исследования: показать эффективность комбинации увлажняющего крема и средства для купания отечественного про изводства (марка «Липобейз baby») в комплексном лечении детей раннего возраста с атопическим дерматитом. Пациенты и методы. В открытое проспективное несравнительное исследование продолжительностью 30 дней включены 30 детей (60% мальчики) в возрасте от 6 до 36 мес со среднетяжелым/легким атопическим дерматитом. На каждом визите (дважды через 2 нед) врач рассчитывал индекс SCORAD и собирал дневники ежедневного учета выраженности зуда кожи и нарушений сна, использования препаратов и нежелательных явлений. Дополнительно оценивали общее впечатление врача от проведенного лечения (индекс GETE). р ( ) Результаты. Средний комплайенс пациентов по использованию увлажняющих препаратов составил 89,2% дней. Средние зна- чения индекса SCORAD снижались по визитам: 23,5±7,3, 18,7±4,9 и 14,6±3,1 балла. Через 2 нед применения увлажняющие средства значительно снижали выраженность зуда (с 6,4 до 3,6 балла) и связанные с ним нарушения сна ребенка (с 2,6 до 1,2 балла). Выявлена прямая умеренная связь (r=0,64; р=0,012) между числом дней без зуда кожи или нарушений сна и компла- йенсом использования увлажняющей косметики. Связанных с препаратами нежелательных явлений не зарегистрировано. Заключение. Включение в комплексную терапию атопического дерматита увлажняющего крема и средства для купания оте- чественного производства (марка «Липобейз baby») повышает эффективность лечения. Ключевые слова: дети, атопический дерматит, эмолиенты, эффективность лечения. Для цитирования: Камаев А.В., Трусова О.В., Бутырина У.А., Макарова И.В. Повышение эффективности лечения детей с атопи- ческим дерматитом в результате использования комплекса эмолиентов. Рос вестн перинатол и педиатр 2019; 64:(3): 109–117. DOI: 10.21508/1027–4065–2019–64–3–109–117 Active studies of atopic dermatitis (AtD) have revealed new disease pathways and confirmed the complex treatment approach need, including different forms of emollients (creams and bath treatments). Active studies of atopic dermatitis (AtD) have revealed new disease pathways and confirmed the complex treatment approach need, including different forms of emollients (creams and bath treatments). Study aim: to show effectiveness of locally produced moisturizing cream and bathing oil combination (Lipobase baby trademark) in the complex treatment of babies and toddlers with AtD. g ( ) Study aim: to show effectiveness of locally produced moisturizing cream and bathing oil combination (Lipobase baby trademark) in the complex treatment of babies and toddlers with AtD. Patients and methods. Thirty children (60% boys) aged from 6 to 36 months with mild/moderate AtD were included in open pro- spective non-comparative study 30 days long. А.В. Камаев1, О.В. Трусова1, У.А. Бутырина2, И.В. Макарова1 А.В. Камаев1, О.В. Трусова1, У.А. Бутырина2, И.В. Макарова1 1ФГБОУ ВО «Первый Санкт-Петербургский государственный медицинский университет им. академика И.П. Павлова» Минздрава России, Санкт-Петербург, Россия; 2Детское поликлиническое отделение №60 Городской поликлиники №91, Санкт-Петербург, Россия A.V. Kamaev1, O.V. Trusova1, U.A. Butyrina2, I.V. Makarova1 A.V. Kamaev1, O.V. Trusova1, U.A. Butyrina2, I.V. Makarova1 A.V. Kamaev1, O.V. Trusova1, U.A. Butyrina2, I.V. Makarova1 1Pavlov First Saint Petersburg State Medical University, Saint Petersburg, Russia; 2P di i i d №60 f Ci i li i №91 S i P b g y g Pediatric outpatient department №60 of City outpatient clinic №91, Saint Petersburg, Russia В ПОМОЩЬ ПРАКТИЧЕСКОМУ ВРАЧУ дерматитом не только дерматологам или аллерголо- гам-иммунологам, но и в первую очередь педиатрам широкого профиля. В отечественной педиатрической практике ранее широко была распространена рекомендация по огра- ничению в купании детей с атопическим дерма- титом [9]; современные руководства и изменившиеся подходы к использованию нелекарственных средств ухода за кожей отвергают это положение [7, 10]. При условии использования прохладных (36–37℃) непродолжительных (до 10 мин) ванн с применением бесщелочных очищающих средств можно не только достичь дополнительной гидратации кожи ребенка, но и обеспечить лучший доступ лечебных и пита- тельных средств для улучшения функций эпидер- миса [11]. Известные попытки первичной профилактики болезней атопического круга пока представляются безуспешными в глобальном плане. Растет распро- страненность не только атопического дерматита, но и аллергического ринита, и бронхиальной астмы у детей [4, 5]. Для аллерголога-иммунолога атопический дер- матит в раннем возрасте может служить маркером повышенного риска формирования аллергического ринита и бронхиальной астмы у ребенка в будущем. Один из наиболее эффективных методов первичной профилактики дерматита и вторичной профилактики последующего движения ребенка по «атопическому маршу» в сторону респираторной аллергии – исполь- зование средств лечебной косметики [6]. Настоящее исследование предпринято в условиях клинической практики для оценки эффективности комплекса средств ухода за кожей отечественного производства: увлажняющего и восстанавливаю- щего крема и масла для купания «Липобейз baby» в ведении пациентов раннего возраста с атопическим дерматитом. По данным, представленным произво- дителем, «Липобейз baby», крем для тела содержит физиологические натуральные масла (макадамии, оливковое, кокосовое, зародышей пшеницы) и обеспечивает кожу комплексом липидов, необхо- димых для восстановления и поддержания кожного барьера, питает и смягчает кожу, делает ее эластичной и гладкой. Другие компоненты крема оказывают увлажняющее (хлопковое молочко), регенерирующее (пантенол) и антиоксидантное (экстракт розмарина и витамин Е) действия. Входящий в состав экстракт розмарина нормализует баланс микробиома кожи. Крем не содержит парабенов, минеральных масел, вазелина. В составе средства «Липобейз baby масло для купания» – масла макадамии, оливковое, мин- дальное и зародышей пшеницы. Эти компоненты бережно очищают кожу, восстанавливая и поддер- живая ее барьерную функцию. Масло поддерживает баланс микробиома кожи благодаря экстракту роз- марина и дает антиоксидантный эффект благодаря витамину Е. Актуальная концепция патогенеза атопического дерматита в настоящее время предполагает равные и взаимодополняющие роли трех ключевых компо- нентов: пищевой сенсибилизации, межклеточных контактов эпидермиса (в частности, состоятель- ности филлагрина) и хронического иммунного воспаления кожи [7, 8]. Исходя из этого все совре- менные руководства подчеркивают необходимость мотивации родителей на регулярное долгосрочное применение комплекса средств ухода за кожей – лечебной косметики наряду с активным использова- нием противовоспалительных средств (топических глюкокортикостероидов и топических ингибиторов кальциневрина). © Коллектив авторов, 2019 Адрес для корреспонденции: Камаев Андрей Вячеславович – к.м.н., доц. кафедры общей врачебной практики (семейной медицины) Первого Санкт-Петербургского государственного медицинского университета им. акад. И.П. Павлова, ORCID: 0000-0001-9654-3429 andykkam@mail.ru Трусова Ольга Валерьевна – к.м.н., доц. кафедры госпитальной терапии с курсом аллергологии и иммунологии им. акад. М.В. Черноруцкого Пер- вого Санкт-Петербургского государственного медицинского университе- та им. акад. И.П. Павлова Макарова Ирина Вадимовна – к.м.н., доц. кафедры госпитальной тера- пии с курсом аллергологии и иммунологии им. акад. М.В. Черноруцкого Первого Санкт-Петербургского государственного медицинского универ- ситета им. акад. И.П. Павлова, гл. внештатный детский аллерголог-имму- нолог Комитета по здравоохранению Санкт-Петербурга 197022 Санкт-Петербург, ул. Льва Толстого, д. 6/8. Бутырина Ульяна Андреевна – врач-педиатр Детского поликлинического отделения №60 Городской поликлиники №91 198329 Санкт-Петербург, просп. Ветеранов, д. 143, корп. 2. В ПОМОЩЬ ПРАКТИЧЕСКОМУ ВРАЧУ Такие средства лечебной косме- тики широко представлены различными производи- телями в аптечной сети; нередко выбор конкретной марки представляет затруднение не только для роди- телей пациента, но и для постоянно наблюдающего ребенка педиатра широкого профиля. Результаты исследований, проведенных с участием российских пациентов, могут служить одним из аргументов в пользу выбора лечебной косметики определенного производителя. Кроме того, представленная работа оценивает безопасность применения обсуждаемой гаммы кос- метических средств и удовлетворенность родителей использованием «Липобейз baby» в комплексном лечении атопического дерматита. © Коллектив авторов, 2019 Адрес для корреспонденции: Камаев Андрей Вячеславович – к.м.н., доц. кафедры общей врачебной практики (семейной медицины) Первого Санкт-Петербургского государственного медицинского университета им. акад. И.П. Павлова, ORCID: 0000-0001-9654-3429 andykkam@mail.ru © Коллектив авторов, 2019 Адрес для корреспонденции: Камаев Андрей Вячеславович – к.м.н., доц. кафедры общей врачебной практики (семейной медицины) Первого Санкт-Петербургского государственного медицинского университета им. акад. И.П. Павлова, ORCID: 0000-0001-9654-3429 andykkam@mail.ru Активные исследования атопического дерматита выявляют новые звенья патогенеза и подтверждают необходимость комплексного подхода к лечению, включающего различные формы выпуска увлажняющих препаратов (крем и средство для купания). Initial and after each 2 weeks SCORAD and dairies for 10 points skin itch and sleep disturbance scores, moisturizers consumption and adverse events were evaluated. Global evaluation of treatment effectiveness score were performed by patient’s doctor. Results. Medium compliance for moisturizers consumption was 89.2% days. Medium SCORAD results decreased from visit 1 to 3: from 23.5±7.3 points to 18.7±4.9 points and to 14.6±3.1 points. After 2 weeks complex therapy including investigated moisturizers, medium day skin itch score was reduced from 6.4 to 3.6 points, also as sleep disturbance score from 2.6 to 1.2 points. Direct mod- erate correlation (r=0.64, p=0.012) was revealed between number of days without skin itch or sleep disturbance and compliance to moisturizers consumption. No any adverse event caused by investigated products was registered. moisturizers consumption. No any adverse event caused by investigated products was registered. Conclusion. Inclusion of locally produced moisturizing cream and bathing oil (Lipobase baby trademark) in the complex AtD therapy improve the treatment efficacy. p y y g p g Inclusion of locally produced moisturizing cream and bathing oil (Lipobase baby trademark) in the complex AtD therapy treatment efficacy. Key words: children,atopic dermatitis, emollients, treatment efficacy. For citation: Kamaev A.V., Trusova O.V., Butyrina U.A., Makarova I.V. Improvement of therapy efficacy in children with atopic dermatitis due to emollient complex. Ros Vestn Perinatol i Pediatr 2019; 64:(3): 109–117 (in Russ). DOI: 10.21508/1027–4065–2019–64–3–109–117 For citation: Kamaev A.V., Trusova O.V., Butyrina U.A., Makarova I.V. Improvement of therapy efficacy in children with atopic dermatitis due to emollient complex. Ros Vestn Perinatol i Pediatr 2019; 64:(3): 109–117 (in Russ). DOI: 10.21508/1027–4065–2019–64–3–109–117 П р м на протяжении ближайших десятилетий отмечается рост распространенности этой нозологии, особенно среди детей первого года жизни [1–3]. Такая распро- страненность прямо определяет актуальность пред- ставления информации об алгоритмах диагностики и ведения педиатрических пациентов с атопическим П роблема ведения пациентов с атопическим дер- матитом привлекает внимание не только дерма- тологов и аллергологов-иммунологов, но и педиатров широкого профиля, а также врачей других специаль- ностей. Как в отечественных, так и в более крупных международных эпидемиологических исследованиях 109 РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) В ПОМОЩЬ ПРАКТИЧЕСКОМУ ВРАЧУ Трусова Ольга Валерьевна – к.м.н., доц. кафедры госпитальной терапии с курсом аллергологии и иммунологии им. акад. М.В. Черноруцкого Пер- вого Санкт-Петербургского государственного медицинского университе- та им. акад. И.П. Павлова Характеристика детей и методы исследования Исследование проведено на базе трех аллерголо- гических кабинетов поликлинической сети г. Санкт- Петербурга: детского поликлинического отде- ления №41 городской поликлиники №44, детского поликлинического отделения №16 городской поли- клиники №114, а также городского аллергокабинета детской городской поликлиники №44. Дизайн протокола – открытое несравнительное проспективное исследование. В работу включены данные 30 пациентов, наблюдавшихся по поводу Бутырина Ульяна Андреевна – врач-педиатр Детского поликлинического отделения №60 Городской поликлиники №91 198329 Санкт-Петербург, просп. Ветеранов, д. 143, корп. 2. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) Камаев А.В. и соавт. Повышение эффективности лечения детей с атопическим дерматитом в результате использования комплекса эмолиентов атопического дерматита в указанных поликли- нических центрах. Продолжительность исследо- вания – 30 дней, на протяжении которых каждый из включенных пациентов осуществил 3 визита с интервалом 14 дней (допускались отклонения от запланированной даты визита не более 2 дней). Препараты «Липобейз baby крем» и «Липобейз baby масло» для купания для пациентов на все время участия в исследовании предоставлены производи- телем – ООО «Фармтек». Частота визитов выбрана для оценки комплайенса в отношении использо- вания средств лечебной косметики, а также для реги- страции возможных нежелательных явлений. Применение увлажняющих препаратов осуществ- лялось согласно рекомендациям производителя: крема не реже чем 2 раза в день ежедневно, а масла для купания – через день для купания в большой ванне или ежедневно при использовании душа. Зуд кожных покровов оценивали по визуальной аналоговой шкале (ВАШ) от 0 до 10 баллов: 0 баллов – отсутствие зуда, от 3 до 5 баллов – слабый зуд, от 6 до 8 баллов – умеренный зуд, от 9 до 10 баллов – тяжелый зуд. Нарушение сна также оценивали по ВАШ от 0 до 10 баллов, где 0 – отсутствует, 10 – сильно выражено. Отдельно учитывали число дней, в которые симптомы атопического дерматита нара- стали (субъективное ухудшение), и число дней, когда пациентам потребовалось внепланово использовать противовоспалительный препарат (топические глю- кокортикостероиды/топические ингибиторы каль- циневрина, в зависимости от тяжести обострения). По данным дневников самонаблюдения, а также по результатам сбора анамнеза на визитах 2 и 3 опре- деляли характер, частоту нежелательных явлений и их возможную связь с применением средств лечебной косметики. Полученные результаты обрабатывали с исполь- зованием пакета StatisticaforWindows 10.0 («Statsoft Inc»., США). Данные представлены в виде сред- него (М) и его стандартной ошибки (±σ), в неко- торых случаях – в виде медианы (Ме) с указанием первого и третьего квартилей [Q25; Q75]. * Здесь и далее процент вычислен условно, так как число детей меньше 100. Характеристика детей и методы исследования Для оценки различий результатов выборок, учитывая вероят- ность отклонений от нормальности распределения, использовали непараметрический U-критерий Манна–Уитни. Различия считали статистически зна- чимыми при р<0,05. При расчете корреляций в связи с оценкой связи между количественными и ранго- выми показателями, а также небольшим объемом выборки использовали коэффициент Спирмена. Для обеспечения соответствия клинической практике при отборе пациентов для участия в про- токоле использован минимум критериев включения и невключения. К критериям включения относились: – – возраст от 6 до 35 мес; – – установленный не менее 1 мес перед включе- нием диагноз атопического дерматита в соответствии Ф [7] – – установленный не менее 1 мес перед включе- нием диагноз атопического дерматита в соответствии с Федеральными клиническими рекомендациями [7]; – – индекс SCORAD от 15 до 60 баллов. с Федеральными клиническими рекомендациями [7]; – – индекс SCORAD от 15 до 60 баллов. Среди критериев невключения: нежелание сотрудничества в рамках протокола (частота визитов, заполнение дневника) у родителей пациента; известные анамнестические реакции на приме- нение компонентов гаммы «Липобейз»; пиодермия или тяжелое, непрерывно-рецидивирующее течение, установленное при осмотре в день визита включения. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) Результаты Четкая связь обострений с употреблением опре- деленного пищевого продукта прослеживалась у 22 РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) В ПОМОЩЬ ПРАКТИЧЕСКОМУ ВРАЧУ (73,3%) детей, у остальных причинами обострений были нарушения работы желудочно-кишечного тракта (как задержки, так и учащение/разжижение стула) – у 5 (16,7%) детей и ОРВИ – у 3 (10%). Среди пациентов с анамнестически очерченной пищевой сенсибилизацией ведущим аллергеном было коровье молоко (у 21 ребенка, или 95,4%), пшеница (у 10 детей, или 45,4%), белок куриного яйца (у 9 детей, или 40,9%) и овсяная крупа (у 7 детей, или 31,8%). Комплайенс в отношении использования пре- паратов (число дней применения «Липобейз baby крема» 2 раза и более, по данным дневников самона- блюдения) составил 100% у 25 (83,3%) детей к визиту 2; у остальных 5 детей он колебался от 85,8 до 92,9%. К визиту 3 доля пациентов, полностью выполнивших назначения врача, составила 80% (24 ребенка); еще 5 детей пропустили от одного до двух дней (комплайенс более 80%), один пациент пропустил 5 дней использо- вания в связи с поездкой к бабушке (препарат забыли дома), однако это не привело к обострению атопиче- ского дерматита. Расход препаратов составил по одной упаковке каждого из средств в течение 12–16 дней, на всю продолжительность исследования – по две упаковки «Липобейз baby крема» и масла для купания на каждого пациента. На визите 1 зарегистрировано обострение атопи- ческого дерматита у 18 (60%) больных, в том числе в подгруппе пациентов со среднетяжелым течением – у 7 (38,9%) и в подгруппе пациентов с легким тече- нием заболевания – у 11 (61,1%). Всем детям, наряду с выдачей препаратов гаммы «Липобейз» назначены топические глюкокортикостероиды или топические ингибиторы кальциневрина в соответствии с обще- принятой клинической практикой в зависимости от объема и выраженности зарегистрированных обост- рений на сроки от 5 до 10 дней. Из 30 детей 12 (40%) были включены в протокол в состоянии ремиссии, им выдавались письменные рекомендации по соче- танному применению средств лечебной косметики («Липобейз baby крем» и «Липобейз baby масло» для купания) и противовоспалительного средства (топические глюкокортикостероиды/топические ингибиторы кальциневрина) с первого дня обост- рения, в случае его развития вне сроков визита по протоколу. Исходно средний по группе индекс SCORAD составил 23,5±7,3 балла. В дальнейшем, к визиту 2 и особенно к визиту 3, средние значения индекса SCORAD неуклонно снижались (рис. 1). Результаты При сопо- ставлении результатов визита 1 и визита 3 оказалось, что снижение индекса SCORAD достигло статисти- ческой значимости (р=0,041). Это прямо связано с уменьшением числа пациентов в состоянии обост- рения дерматита. Данные по разделению пациентов на подгруппы легкого и среднетяжелого течения атопического дерматита, а также по числу пациентов в состоянии обострения на каждый из визитов исследования и средним значениям индекса SCORAD (±σ) по более мелким подгруппам представлены в сводной табл. 1. Все пациенты, включенные в исследование, осу- ществили оба визита последующего наблюдения в запланированные протоколом сроки; анализи- руемая выборка для каждого из визитов составляет 30 пациентов. Как по суммарному индексу SCORAD, так и по отдельным субъективным показателям наиболее значимым для качества жизни ребенка с атопиче- ским дерматитом и членов его семьи – зуду кожи и нарушению сна – наблюдалась отчетливая поло- жительная динамика. Поскольку в отличие от харак- тера и площади поражения кожи оба эти показателя всегда оцениваются родителями, в рамках настоя- щего исследования мы включили эти показатели в дневник самонаблюдения для ежедневной оценки. На рис. 2 представлены средние значения обоих показателей с недельными интервалами; статисти- чески значимое снижение на фоне применения средств лечебной косметики «Липобейз baby крем» К визиту 2 число пациентов, у которых зареги- стрировано обострение атопического дерматита, уменьшилось до 7 (23,3%), в их числе: 5 (71,4%) из 19 детей со среднетяжелым течением и 2 (28,6%) из 11 – с легким течением болезни. К визиту 3 в состоянии обострения дерматита находились только 3 ребенка со среднетяжелым течением забо- левания, у 2 из них это обострение было спрово- цировано погрешностью в элиминационной диете в предшествовавшую визиту неделю – контактом с известным пищевым аллергеном. Таблица 1. Распределение пациентов по степени тяжести атопического дерматита, фазам обострения и ремиссии по визи- там, а также значения индекса SCORAD в подгруппах Table 1 Atopic dermatitis severity exacerbation or remission phase distribution by visits and SCORAD results in subgroups Таблица 1. Распределение пациентов по степени тяжести атопического дерматита, фазам обострения и ремиссии по визи- там, а также значения индекса SCORAD в подгруппах 112 РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) Table 1. Результаты В качестве объективного инструмента оценки динамики патологического процесса при атопи- ческом дерматите использовали индекс SCORAD, расчет которого проводили при включении в иссле- дование и затем на каждом визите пациента в центр [12]. Кроме того, на визитах 2 и 3 наблю- дающий врач аллерголог-иммунолог осуществлял общую оценку эффективности терапии (GETE index) в градациях оценки от «ухудшения», «состояния без перемен», до «улучшения» либо «выраженного улучшения» состояния пациента. К «улучшению» относили сохранение сухости кожи и высыпания при уменьшении их интенсивности и площади; к «выраженному улучшению» – отсутствие жалоб и объективных находок при осмотре. В качестве объективного инструмента оценки динамики патологического процесса при атопи- ческом дерматите использовали индекс SCORAD, расчет которого проводили при включении в иссле- дование и затем на каждом визите пациента в центр [12]. Кроме того, на визитах 2 и 3 наблю- дающий врач аллерголог-иммунолог осуществлял общую оценку эффективности терапии (GETE index) в градациях оценки от «ухудшения», «состояния без перемен», до «улучшения» либо «выраженного улучшения» состояния пациента. К «улучшению» относили сохранение сухости кожи и высыпания при уменьшении их интенсивности и площади; к «выраженному улучшению» – отсутствие жалоб и объективных находок при осмотре. В исследование включены 30 пациентов, в том числе 18 (60%)* мальчиков. Медиана возраста в выборке составила 13,6 мес, первый и третий квар- тили ([Q25; Q75]) – [7,2; 19,5] мес соответственно. Давность регистрации диагноза атопического дерма- тита преимущественно превышала 10 мес: медиана 10,6 [5,8; 15,4] мес. Подгруппа легкого течения атопического дерма- тита состояла из 19 (63,3%) детей, подгруппа средне- тяжелого течения – из 11 (36,7%). Для купирования обострений основным противовоспалительным сред- ством были топические глюкокортикостероиды у 8 (26,7%) пациентов, топические ингибиторы кальци- неврина – у 15 (50%), у 7 (23,3%) детей обострения купировали без использования противовоспали- тельных средств (пероральные антигистаминные средства, препараты цинка, увеличение объема эмо- лиентов). Кроме индексов SCORAD и GETE, учитывали данные ежедневной субъективной оценки зуда кожи и нарушения сна из-за зуда кожи, отмечав- шиеся родителями ежедневно в дневнике пациента. Для объективизации динамики у каждого пациента по данным показателям рассчитывали среднее зна- чение на 1–4-й неделях, а затем полученные данные компоновали по всем включенным пациентам. Результаты Atopic dermatitis severity, exacerbation or remission phase distribution by visits and SCORAD results in subgroups Течение атопического дерматита Фаза атопического дерматита Визит 1 Визит 2 Визит 3 n SCORAD, баллы n SCORAD, баллы n SCORAD, баллы Легкое (n=19) Обострение 11 22,6±3,1 2 23,2±1,8 0 – Ремиссия 8 14,1±1,7 17 13,5±2,9 19 9,8±1,4 Среднетяжелое (n=11) Обострение 7 37,7±5,6 5 35,9±3,7 3 40,6±5,9 Ремиссия 4 19,8±4,2 6 17,4±2,1 8 16,3±2,3 rity, exacerbation or remission phase distribution by visits and SCORAD results in subgroups Table 1. Atopic dermatitis severity, exacerbation or remission phase distribution by visits and SCORAD results in РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) Камаев А.В. и соавт. Повышение эффективности лечения детей с атопическим дерматитом в результате использования комплекса эмолиентов и «Липобейз baby масло» для купания наблюдалось уже после 2 нед использования препаратов. и комплайенса в отношении лечебной косметики по 3 рангам: полный, удовлетворительный (>80%) и неудовлетворительный (≤80%) для всей выборки в целом получена умеренная прямая связь (r=0,64; р=0,012). Максимальной выраженности корреляция выявлена между показателями «использование масла для купания» (3 ранга: «ежедневное», «по рекомен- дациям (через день)» и «реже чем рекомендовано») и балльной оценкой зуда кожи – сильная обратная связь (r=–0,82; р=0,0001). Более подробно различные корреляции, выявленные в ходе настоящего исследо- вания, представлены в табл. 3. Число дней, в которые пациенты не испытывали зуд кожи и/или нарушения сна, также значительно увеличивалось на протяжении регулярного приме- нения средств лечебной косметики; лучших резуль- татов здесь ожидаемо достигла подгруппа пациентов с легким течением заболевания, включенных в иссле- дование вне стадии обострения. Более подробно данные представлены в табл. 2. При расчете корреляции показателей «коли- чество дней без зуда кожи либо нарушения сна» ИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) 23,5 18,7 14,6 0 8 16 24 32 Визит 1 Визит 2 Визит 3 * Рис. 1. Динамика средних значений индекса SCORAD (±σ) по визитам, баллы. * – р=0,041. Fig. 1. Mean (±σ) SCORAD readings change by visits, points. * – р=0,041. 0 1 2 3 4 5 6 7 8 Исходно 1-я неделя 2-я неделя 3-я неделя 4-я неделя Зуд кожи Нарушение сна Рис. 2. Уменьшение средней балльной оценки выраженности зуда кожи и нарушений сна у па- циентов по неделям наблюдения. Fig. 2. Mean itch intensity and sleep disturbance score decrease by weeks of observation. 23,5 18,7 14,6 0 8 16 24 32 Визит 1 Визит 2 Визит 3 * Рис. Результаты Correlation analysis of emollient utilization compliance and skin itch or sleep disturbance intensity Параметр Комплайенс по крему по маслу для купания общий по препаратам Выраженность зуда r=–0,54 (р=0,06) r=–0,82 (р=0,0001) r=–0,59 (р=0,04) Нарушения сна r=–0,37 (р=0,18) r=–0,12 (р=0,8) r=–0,26 (р=0,03) Число дней без зуда кожи или нарушений сна r=0,62 (р=0,026) r=0,41 (р=0,052) r=0,64 (р=0,012) Таблица 3. Корреляционный анализ использования препаратов лечебной косметики гаммы «Липобейз baby» и интенсивно- сти зуда и нарушений сна у детей Table 3. Correlation analysis of emollient utilization compliance and skin itch or sleep disturbance intensity Параметр Комплайенс по крему по маслу для купания общий по препаратам Выраженность зуда r=–0,54 (р=0,06) r=–0,82 (р=0,0001) r=–0,59 (р=0,04) Нарушения сна r=–0,37 (р=0,18) r=–0,12 (р=0,8) r=–0,26 (р=0,03) Число дней без зуда кожи или нарушений сна r=0,62 (р=0,026) r=0,41 (р=0,052) r=0,64 (р=0,012) Таблица 3. Корреляционный анализ использования препаратов лечебной косметики гаммы «Липобейз baby» и интенсивно- сти зуда и нарушений сна у детей Table 3. Correlation analysis of emollient utilization compliance and skin itch or sleep disturbance intensity Параметр Комплайенс по крему по маслу для купания общий по препаратам Выраженность зуда r=–0,54 (р=0,06) r=–0,82 (р=0,0001) r=–0,59 (р=0,04) Н 0 37 ( 0 18) 0 12 ( 0 8) 0 26 ( 0 03) Таблица 3. Корреляционный анализ использования препаратов лечебной косметики гаммы «Липобейз baby» и интенсивно- сти зуда и нарушений сна у детей Table 3. Correlation analysis of emollient utilization compliance and skin itch or sleep disturbance intensity рреляционный анализ использования препаратов лечебной косметики гаммы «Липобейз baby» и интенсивн ушений сна у детей ti l i f lli t tili ti li d ki it h l di t b i t it Как видно из данных, представленных в табл. 3, применение препаратов для восполнения липидов эпидермиса, восстановления барьерной функции кожи и ее дополнительного увлажнения умень- шает выраженность зуда кожи, нарушения сна у детей с атопическим дерматитом и повышает сте- пень контроля заболевания в целом. Наблюдающие врачи отмечали только «улучшение» (8 пациентов, или 26,7%) либо «выраженное улучшение» (22 паци- ента, или 73,3%) в состоянии включенных пациентов. Даже у пациентов, у которых произошло обострение к моменту визита 3 (3 человека из группы среднетяже- лого дерматита), общее впечатление от курса лечения в течение 30 дней расценено как «улучшение». доступной гаммы средств отечественного производ- ства в целях лечебного ухода за атопичной кожей, что может быть дополнительным мотивирующим фактором для отдельных пациентов при выборе лечебного комплекса [10, 15]. Результаты Для пациентов с легким/среднетяжелым течением атопического дер- матита в дошкольном возрасте расход увлажняющего крема в количестве 1 упаковки за 2 нед может быть признан минимально достаточным; у некоторых более крупных детей или при более выраженных про- явлениях можно рекомендовать и более интенсивное использование, до 1 упаковки в неделю [7, 15]. Представляется интересным, что независимо от ключевого триггера обострений даже у пациентов без пищевой сенсибилизации была показана эффек- тивность комплекса увлажняющих препаратов «Липо- бейз baby» (крем и масло для купания). Понимание наличия разных фенотипов и клинико-патогенетиче- ских вариантов атопического дерматита активно обсу- ждается в актуальной медицинской периодике, однако подчеркивается, что базовый уход за кожей необходим всем пациентам, независимо от ведущего патогенети- ческого звена атопического дерматита [16, 17]. Не отмечено нежелательных реакций, связанных с использованием увлажняющего крема или масла для купания «Липобейз baby», на протяжении 30 дней участия в протоколе. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) Результаты 1. Динамика средних значений индекса SCORAD (±σ) по визитам, баллы. * – р=0,041. Fig. 1. Mean (±σ) SCORAD readings change by visits, points. * – р=0,041. Рис. 1. Динамика средних значений индекса SCORAD (±σ) по визитам, баллы. * – р=0,041. р , Fig. 1. Mean (±σ) SCORAD readings change by visits, points. * – р=0,041. Й Й 0 1 2 3 4 5 6 7 8 Исходно 1-я неделя 2-я неделя 3-я неделя 4-я неделя Зуд кожи Нарушение сна Рис. 2. Уменьшение средней балльной оценки выраженности зуда кожи и нарушений сна у па- циентов по неделям наблюдения. Fig. 2. Mean itch intensity and sleep disturbance score decrease by weeks of observation. 0 1 2 3 4 5 6 7 8 Исходно 1-я неделя 2-я неделя 3-я неделя 4-я неделя Зуд кожи Нарушение сна Рис. 2. Уменьшение средней балльной оценки выраженности зуда кожи и нарушений сна у па- циентов по неделям наблюдения. Fig. 2. Mean itch intensity and sleep disturbance score decrease by weeks of observation. Рис. 2. Уменьшение средней балльной оценки выраженности зуда кожи и нарушений сна у па- циентов по неделям наблюдения. Fig. 2. Mean itch intensity and sleep disturbance score decrease by weeks of observation. Fig. 2. Mean itch intensity and sleep disturbance score decrease by weeks of observation. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) В ПОМОЩЬ ПРАКТИЧЕСКОМУ ВРАЧУ Таблица 2. Среднее число дней (±σ) без зуда кожи и нарушений сна в подгруппах по неделям лечения Table 2. Mean (±σ) number of days without skin itch and sleep disturbance by weeks of treatment Таблица 2. Среднее число дней (±σ) без зуда кожи и нарушений сна в подгруппах по неделям лечения Table 2. Mean (±σ) number of days without skin itch and sleep disturbance by weeks of treatment Течение атопического дерматита Фаза заболевания Неделя 1-я 2-я 3-я 4-я Легкое Обострение 2,1±0,9 3,5±1,1 4,7±0,6 5,4±1,2 Ремиссия 5,4±1,3 6,1±0,9 6,3±1,5 6,7±0,5 Среднетяжелое Обострение 0,8±0,7 1,7±0,6 3,6±0,8 3,8±0,4 Ремиссия 4,3±1,1 4,5±1,2 5,1±0,7 5,2±1,2 Таблица 3. Корреляционный анализ использования препаратов лечебной косметики гаммы «Липобейз baby» и интенсивно- сти зуда и нарушений сна у детей Table 3. Обсуждение Потребность в регулярном, долгосрочном исполь- зовании средств лечебной косметики пациентами с атопическим дерматитом как один из ключевых компонентов достижения контроля и удержания ремиссии в настоящее время не вызывает сомнения у практикующих дерматологов и аллергологов [13, 14]. В то же время для пациентов и их родителей особенно важно мнение постоянно наблюдающего педиатра широкого профиля, а не только врачей- консультантов. Вот почему для сохранения прием- лемого уровня комплайенса необходимо знакомить широкую педиатрическую аудиторию с новыми исследованиями по оценке эффективности исполь- зования лечебной косметики у детей с атопическим дерматитом. Настоящую работу отличает применение В представленной работе показано повышение эффективности терапии атопического дерматита при использовании комплекса средств увлажнения кожи. Важно, что у отдельных пациентов применение этого комплекса начато в фазу обострения заболе- вания. Вопрос о сроках назначения лечебной косме- тики и возможности ее сочетания с фармакологиче- скими противовоспалительными лекарственными препаратами нередко встает перед практикующими врачами. Дополнительное подтверждение безопас- ности и эффективности раннего назначения увлаж- няющих средств отвечает требованиям современных РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) Камаев А.В. и соавт. Повышение эффективности лечения детей с атопическим дерматитом в результате использования комплекса эмолиентов клинических рекомендаций [7, 8] и будет способство- вать внедрению такого подхода в широкую клиниче- скую практику. Гамма «Липобейз baby» эффективна для контроля обоих субъективных показателей течения дерматита. обоих субъективных показателей течения дерматита. В целом средства лечебной косметики «Липобейз baby крем» и масло для купания показали высокую эффективность в достижении и удержании ремиссии у детей раннего возраста (0,5–3 лет жизни) с ато- пическим дерматитом легкого и среднетяжелого течения при начале терапии как в фазу обострения, так и ремиссии заболевания. Показана высокая удо- влетворенность наблюдающих врачей и родителей пациентов проводимым лечением. Не зарегистри- ровано случаев нежелательных явлений либо непе- реносимости, связанных с использованием гаммы «Липобейз baby». Полученные в настоящем прото- коле результаты в отношении скорости достижения ремиссии у больных атопическим дерматитом, динамики индекса SCORAD и показатели удовле- творенности пациентов и наблюдающих врачей сопоставимы с результатами апробаций немецких и французских косметических средств, ранее прово- дившихся авторами [18, 19]. ЛИТЕРАТУРА (REFERENCES) 8. Werfel T., Heratizadeh A., Aberer W., Ahrens F., Augustin M., Biedermann T. et al. S2k guideline on diagnosis and treatment of atopic dermatitis – short version. Allergo J Int 2016; 25: 82–95. DOI: 10.1007/s40629-016-0110-8 1. Смирнова Г.И. Актуальный атопический дерматит: про- блемы и перспективы. Российский аллергологический журнал 2017; 14: 4–5: 30–39. [Smirnova G.I. Actual atopic dermatitis: problemsand perspectives. Rossiiskii allergo- logicheskii zhurnal 2017; 14: 4–5: 30–39. (in Russ)] 9. Скрипкин Ю. К., Зверькова Ф.А., Шарапова Г.Я., Студ- ницын А.А. Руководство по детской дерматовенероло- гии. Ленинград: Медицина, 1983; 477. [Skripkin Yu. K., Zver’kova F.A., Sharapova G.Ja., Studnicyn A.A. Pediatric dermatovenerology guideline. Lenigrad: Meditsina, 1983; 477. (in Russ)] 2. Mortz C.G., Andersen K.E., Dellgren C., Barington T., Bindslev- Jensen C. Atopic dermatitis from adolescence to adulthood in the TOACS cohort: prevalence, persistence and comorbidi- ties. Allergy 2015; 70(7): 836–845. DOI: 10.1111/all.12619 3. Al-naqeeb J., Danner S., Fagnan L.J., Ramsey K., Michaels L.A., Mitchell J. et al. The Burden of Childhood Atopic Dermatitis in the Primary Care Setting: A Report from the Meta-LARC Consortium. J Am Board Fam Med 2019; 32(2): 191–200. DOI: 10.3122/jabfm.2019.02.180225 10. Зайцева С.В., Застрожина А.К., Муртазаева О.А. Наруж- ная терапия атопического дерматита у детей. Медицин- ский совет 2017; 19: 149–152. [Zajceva S.V., Zastrozhi- na A.K., Murtazaeva O.A. Topical therapy of atopic dermatitis in children. Medicinskii sovet 2017; 19: 149–152. (in Russ)] 4. Mallol J., Crane J., von Mutius E., Odhiambo J., Keil U., Stew- art A.; ISAAC Phase Three Study Group. The International Study of Asthma and Allergies in Childhood (ISAAC) Phase Three: a global synthesis. Allergol Immunopathol (Madr) 2013; 41(2): 73–85. DOI: 10.1016/j.aller.2012.03.001 11. Смирнова Г.И. Атопический дерматит у детей: новое в па- тогенезе, диагностике и лечении. Лечащий врач 2017; 4: 12–18. [Smirnova G.I. Atopic dermatitis in children: news in pathogenesis, diagnostics and treatment. Lechashchii vrach 2017; 4: 12–18. (in Russ)] 5. Батожаргалова Б.Ц., Мизерницкий Ю.Л., Подольная М.А. Метаанализ распространенности астмоподобных сим- птомов и бронхиальной астмы в России (по результатам программы ISAAC). Российский вестник перинатоло- гии и педиатрии 2016; 61(4): 59–69. [Batozhargalova B.C., Mizernickij Yu.L., Podol’naja M.A. Metanalysis of the preva- lence of asthma-like symptoms and bronchial asthma in Rus- sia (ISAAC program results). Rossiyskiy Vestnik Perinatologii i Pediatrii (Russian Bulletin of Perinatology and Pediatrics) 2016; 61(4): 59–69. (in Russ)]. DOI: 10.21508/1027-4065- 2016-61-4-59-69 12. Severity scoring of atopic dermatitis: the SCORAD index. Consensus Report of the European Task Force on Atopic Dermatitis. Dermatol 1993; 186(1): 23–31. 13. Обсуждение Наши данные по положительной динамике клю- чевого объективного показателя контроля за тече- нием атопического дерматита – индекса SCORAD – сопоставимы с результатами других работ [10, 14, 15] и подтверждают возможность рекомендации линейки «Липобейз baby крем» и «Липобейз baby масло» для купания для эффективного ведения детей с ато- пическим дерматитом как на этапе обострения, так и ремиссии основного заболевания. Показатели зуда и нарушения сна выбраны как в наибольшей степени ухудшающие качество жизни компоненты патогенеза атопического дерматита у пациентов первых 3 лет жизни. Кроме того, показано, что неукротимый зуд, особенно, ведущий к нарушению сна, существенно влияет на психомоторное развитие детей [9, 11]. Купирование и долгосрочный контроль этого показа- теля меняют течение атопического дерматита и вос- приятие этого заболевания пациентом и родителями. ЛИТЕРАТУРА (REFERENCES) Weidinger S., Novak N. Atopic dermatitis. Lancet 2016; 387:1109. 14. Балаболкин И.И., Булгакова В.А., Елисеева Т.И. Наруше- ние кожного барьера и продукты лечебной косметики для ухода за кожей при атопическом дерматите у де- тей. Фарматека 2018; S1: 64–71. [Balabolkin I.I., Bulga- kova V.A., Eliseeva T.I. Skin barrier impairment and medical cosmetic skin care products for atopic dermatitis in children. Pharmateca 2018; S1: 64–71. (in Russ)] 6. Simpson E.L., Chalmers J.R., Hanifin J.M., Thomas K.S., Cork M.J., McLean W.H. et al. Emollient enhancement of the skin barrier from birth offers effective atopic dermatitis pre- vention. J Allergy Clin Immunol 2014; 134: 818–823. DOI: 10.1016/j.jaci.2014.08.005 15. Камаев А.В., Трусова О.В., Ляшенко Н.Л., Макарова И.В. Эмоленты в терапии атопического дерматита у детей: анализ клинической и фармакоэкономической эф- фективности. Российский аллергологический журнал 2017; 14(4–5): 98–107. [Kamaev A.V., Trusova O.V., Lja- shenko N.L., Makarova I.V. Emolients in atopic dermatitis treatment in children: analysis of clinical and pharmacoeco- 7. Атопический дерматит у детей. Федеральные клини- ческие рекомендации. М., 2016; 60. [Atopic dermatitis in children. Federal clinical recommendations. Moscow, 2016; 60. (in Russ)] РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3) В ПОМОЩЬ ПРАКТИЧЕСКОМУ ВРАЧУ 18. Макарова И.В. Уход за кожей больных атопическим дер- матитом. Аллергология 2005; 1: 47–52. [Makarova I.V. Skin care for atopic dermatitis patients. Аllergologiya 2005; 1: 47–52. (in Russ)] nomical efficacy. Rossiiskii allergologicheskii zhurnal 2017; 14(4–5): 98–107. (in Russ)] nomical efficacy. Rossiiskii allergologicheskii zhurnal 2017; 14(4–5): 98–107. (in Russ)] 16. Cabanillas B., Brehler A.C., Novak N. Atopic dermatitis phe- notypes and the needfor personalized medicine. Curr Opin Allergy Clin Immunol 2017; 17: 309–315. DOI: 10.1097/ ACI.0000000000000376 19. Макарова И.В. Комплексный подход к наружной тера- пии детей с атопическим дерматитом с использованием средств ухода за кожей серии Бальнеум. Вопросы прак- тической педиатрии 2007; 2(6): 60–64. [Makarova I.V. Complex approach to the topic therapy in children with atopic dermatitis: Balneum skin care products usage. Voprosy prakticheskoi pediatrii 2007; 2(6): 60–64. (in Russ)] 17. Roduit C., Frei R., Depner M., Karvonen A.M., Renz H., Braun-Fahrländer C. et al. Phenotypes of Atopic Dermatitis Depending on the Timing of Onset and Progression in Child- hood. JAMA Pediatr 2017; 171(7): 655–662. DOI: 10.1001/ jamapediatrics.2017.0556 Конфликт интересов: Conflict of interest: Products of medical cosmetics for use in this Protocol are provided by the Pharmatek Company LLC. Representatives of the Company did not participate in the statistical processing, interpretation of the results and preparation of this publication. Препараты лечебной косметики для использования в настоящем протоколе предоставлены производите- лем ООО «Фармтек». В статистической обработке, интерпретации результатов и подготовке настоящей публикации представители компании-производителя участия не принимали. The authors of this article confirmed the lack of conflict of interest and financial support, which should be reported. Авторы данной статьи подтвердили отсутствие кон- фликта интересов и финансовой поддержки, о которых необходимо сообщить. Благодарность: Авторы выражают благодарность врачам-аллергологам Беляевой Галине Владимировне, Жиглинской Ольге Владимировне, Медведевой Елене Александровне, Ляшенко Наталье Леонидовне и Осолодкиной Анне Анатольевне за помощь в сборе фактического материала для данного исследования. РОССИЙСКИЙ ВЕСТНИК ПЕРИНАТОЛОГИИ И ПЕДИАТРИИ, 2019; 64:(3) ROSSIYSKIY VESTNIK PERINATOLOGII I PEDIATRII, 2019; 64:(3)
https://openalex.org/W4243268735	https://www.researchsquare.com/article/rs-48802/v1.pdf	English	null	Immunohistochemical Expression of β-catenin, Ki67, CD3 and CD18 in Canine Colorectal Adenomas and Adenocarcinomas	Research Square (Research Square)	2,020	cc-by	5,843	Immunohistochemical Expression of β-catenin, Ki67, CD3 and CD18 in Canine Colorectal Adenomas and Adenocarcinomas Kristin Marie Valand Herstad (  kristin.herstad@nmbu.no ) Norwegian University of Life Sciences (NMBU) https://orcid.org/0000-0003-3942-8584 Gjermund Gunnes Norwegian University of Life Sciences (NMBU) Runa Rørtveit NMBUNorwegian University of Life Sciences (NMBU) Øyvor Kolbjørnsen Norwegian Veterinary Institute Linh Tran Norwegian veterinary institute Ellen Skancke Norwegian University of Life Sciences (NMBU) Research article Keywords: canine, colorectal adenoma and adenocarcinoma, markers for tumour progression, tumour- infiltrating immune-cells Posted Date: August 4th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-48802/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 on March 12th, 2021. See the published version at https://doi.org/10.1186/s12917-021-02829-6. Research article Posted Date: August 4th, 2020 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on March 12th, 2021. See the published version at https://doi.org/10.1186/s12917-021-02829-6. Page 1/17 Abstract Background Inflammation is believed to influence the human colorectal carcinogenesis and may have impact upon prognosis and survival. High presence of tumour-infiltrating CD3+ T-cells, is associated with a better outcome in humans with colorectal cancer. The mucosal immunophenotype in dogs with colorectal cancer is poorly described. The aim of this study was to investigate whether the density, distribution and grade of tumour-infiltrating immune cells (TIIs) in canine colorectal tumours is associated with histologic indicators of malignancy and can be considered a prognostic factor in dogs. This retrospective case-control study was performed on formalin-fixed, paraffin-embedded tissue samples from dogs with histologically confirmed colorectal adenoma (n=18) and adenocarcinoma (n=5) collected from archived samples. The samples had been collected by colonoscopy, surgery or during postmortem examination. Healthy colonic tissue obtained post mortem from dogs euthanized of reasons not involving the gastrointestinal tract, served as control tissue (n=9). Results: The tumour samples had significantly lower numbers of CD3+ T- cells in the epithelium compartment (Wilcoxon test, p=0,0006), as well as significantly lower number of CD18+ cells in the lamina propria, compared to control samples (Wilcoxon test, p=0,001). The Ki67 positive cells showed a strong signal in adenomas and adenocarcinomas. There was no clear distinction with regards to expression levels of the markers for tumour progression (β-catenin, and Ki67) between adenomas and adenocarcinomas. Colonic samples from control dogs had uniform staining of β-catenin along the cell membrane of epithelial cells. When compared to normal colonic cells, the expression levels of cytoplasmic β-catenin were significantly higher in adenomas and adenocarcinomas (Wilcoxon test, p=0,0002). None of the control samples showed positive staining of β-catenin in the nucleus of colonic cells. In contrast, adenocarcinoma and adenoma showed moderate to strong staining of the cell nucleus. Conclusions: β-catenin and Ki67 were not useful markers in distinguishing adenomas from adenocarcinomas. The lower presence of CD18- and CD3+ cells in tumours compared to controls, indicates a reduced presence of histiocytes and T-cells which may have implications for the defense against cancer progression. Materials And Methods This retrospective case-control study was performed on archived formalin-fixed paraffin-embedded tissue samples submitted to NMBU during the period from 1998 to 2015. These samples had been collected for clinical purposes and leftover samples were archived. Owner consent for using leftover samples for research in cancer diseases was given by dog-owners. These samples had been taken for clinical purposes and leftover samples were archived and used for this study. Inclusion criteria for this study were colorectal tissue from dogs with histologically confirmed colorectal adenoma or adenocarcinoma, and 18 adenoma and five adenocarcinomas were included in addition to nine control samples. Colonic tissue from control dogs were collected at necropsy from dogs euthanized for reasons that did not involve the gastrointestinal tract (n = 9). Introduction Colorectal cancer (CRC) made up by adenocarcinoma is one of the most common types of cancer in humans (1). In contrast, dogs are rarely diagnosed with colorectal cancer, albeit more frequently than other mammals (2, 3). This disease is associated with serious clinical signs and has a poor prognosis due to local recurrence and formation of metastases in both humans (4, 5) and dogs (6–8). Sporadic CRC in humans is believed to originate from adenomas through a process of multiple genetic and molecular events (9). Studies in dogs indicate that a similar process occurs in canine colorectal carcinogenesis (10–14). Page 2/17 Page 2/17 Colonic stem cells are located at the bottom of the colonic crypt where they proliferate and migrate towards the top, resulting in mature cells without the capacity to divide. This proliferation process is tightly regulated through the wingless-related integration site (Wnt) signalling pathway and involves the protein β-catenin (15). Mutation of β-catenin is a key factor in the tumorigenesis, as described in humans (15) as well as in dogs (10, 16, 17). A failure in this mechanism results in colonic cells with increased proliferation capacity. The abnormal cellular proliferation along the tumorigenesis may be evaluated by Ki67 (18). The Ki67-protein is only present during active phases of the cell cycle, thus expression of this antigen indicates cell-growth (19). Inflammatory cells have been proposed to play a role in the colorectal carcinogenesis in humans (20, 21), and chronic inflammation such as ulcerative colitis may progress into cancer (22). Colorectal adenomas and adenocarcinomas in humans are infiltrated by inflammatory cells, which may influence the capability of the tumour to proliferate and metastasize (23). The presence of tumour-infiltrating CD3 + T- lymphocytes in human CRC correlates with prognosis, as patients with high tumour infiltration have longer survival than those with poorly infiltrated tumours (24). A study of Japanese miniature-dachshunds showed that the progression from inflammatory polyps into adenoma and carcinoma involves dysregulation of β-catenin (17). However, the inflammation and presence of T-lymphocytes associated with the canine colorectal tumour progression have not been described at the different disease stages. In this work, we characterized and quantified the infiltration of immune cells in canine colorectal adenoma, adenocarcinoma, and normal colonic tissue by using the antigens CD18 and CD3 labelling histiocytes and T-cells, respectively. Evaluation of tumor progression was performed using the antigens β-catenin and Ki67. Selection of cases and control dogs For each case, information about the breed, gender, age, histopathological diagnosis, tumour localization, sampling technique and treatment were obtained from the clinical record (Table 1). The dogs with colorectal adenomas and adenocarcinomas were of various breeds represented by both genders and Page 3/17 Page 3/17 were between 1 and 14 years old, with an average of 8 years. From these dogs, colorectal mucosal samples were collected during surgery (n = 9), colonoscopy (n = 8), or necropsy (n = 2), and in four cases by unknown procedure (Table 1). Page 4/17 Table 1 Overview of dogs and samples Dog no. Breed Gender Age (y) Diagnosis* Tumour location (C/R) Method of sampling Treatment 1 German shephard M 9 Adeno- carcinoma R Surgery Surgery 2 Irish Setter F 10 Adeno- carcinoma R Colonoscopy Surgery 3 Shetland sheepdog M 14 Adeno- carcinoma C Post mortem Meloxicam 4 English springer spaniel M 8 Adeno- carcinoma R Colonoscopy Piroxicam 5 Tibetanian spaniel M/N 10 Adeno- carcinoma R Post mortem Meloxicam 6 German shepherd F 9 Adenoma UN UN UN 7 Irish Setter M 6 Adenoma UN UN UN 8 English setter M 8 Adenoma UN UN UN 9 Mixed breed M 10 Adenoma R Surgery UN 10 German shephard M 4 Adenoma R Surgery Surgery 11 Staffordshire bullterrier M 8 Adenoma R Surgery Surgery 12 Papillon M 10 Adenoma R Surgery Surgery 13 Colli shorthaired M 3 Adenoma R Surgery Surgery 14 Norwegian lundehund M 7 Adenoma R Colonoscopy Surgery The diagnosis was not determined for all control dogs, thus symptoms/syndromes are described in Table 1 Overview of dogs and samples Page 5/17 Dog no. Tissue samples Tissue specimens were fixed in 4% neutral buffered formalin, processed routinely, embedded in paraffin wax, cut into 4 µm thick sections and stained with haematoxylin and eosin (HE). In total, 12 specimens from tumorous tissue were of full thickness, while in 11 specimens, the muscularis mucosae were lacking. The histopathological diagnoses were evaluated by a board-certified veterinary pathologist (GG), according to the guidelines for classification of canine colorectal adenoma and adenocarcinomas (25). These guidelines suggest that tumours are classified as adenocarcinoma only if neoplastic cells invade muscularis mucosa. Although some of our tumours consisted of cellular features strongly indicating malignancy, they were still classified as adenoma if no invasion of basal lamina was found. (25). Selection of cases and control dogs Breed Gender Age (y) Diagnosis Tumour location (C/R) Method of sampling Treatment 15 Cocker spaniel F 10 Adenoma C Colonoscopy no 16 Golden retriever M 2 Adenoma R Surgery Surgery 17 Bichon havanais M 5 Adenoma R Colonoscopy Surgery 18 English setter M 11 Adenoma R Surgery Surgery 19 Gordon setter F 10 Adenoma R Surgery Surgery 20 Grand danois M 10 Adenoma C Colonoscopy Piroxicam 21 Cocker spaniel M 12 Adenoma C Colonoscopy no 22 Border collie F 12 Adenoma R Colonoscopy Surgery 23 English setter F 8 Adenoma UN UN UN 24 West highland white terrier M 15 Respiratory distress NA Post mortem NA 25 Miniatur pincher F 12 Lung tumour NA Post mortem NA 26 Staffordshire bullterrier F 13 General weakness NA Post mortem NA 27 French bulldog M 3 Intervertebral disk hernia NA Post mortem NA 28 Alaskan malamute F 7 Polyneuropathy NA Post mortem NA 39 French bullldog F 3 Degenerative disk disease NA Post mortem NA 30 Collie, longhair M UN Epilepsy NA Post mortem NA The diagnosis was not determined for all control dogs, thus symptoms/syndromes are described in some of the cases Page 6/17 The diagnosis was not determined for all control dogs, thus symptoms/syndromes are described in some of the cases. UN, unkown NA, not applicable RTA, road traffic accident Page 6/17 The diagnosis was not determined for all control dogs, thus symptoms/syndromes are described in some of the cases. UN, unkown NA, not applicable RTA, road traffic accident Page 6/17 Dog no. Breed Gender Age (y) Diagnosis Tumour location (C/R) Method of sampling Treatment 31 Pug dog M 5 Urolithiasis NA Post mortem NA 32 Chihuahua M/N 3 Multiple fractures, RTA NA Post mortem NA The diagnosis was not determined for all control dogs, thus symptoms/syndromes are described in some of the cases. UN, unkown NA, not applicable RTA, road traffic accident The control dogs consisted of various breeds from both genders, and were between 3 and 15 years old, with an average age of 8 years (Table 1). Immunohistochemistry The following antibodies were used; mouse anti-β-catenin (BD Biosciences, Franklin Lakes, New Jersey), rabbit-anti-CD3 (DAKO, A 0452 North America Inc, California), mouse-anti-dog-CD18 (Leucocyte Antigen Laboratory, California) and anti-Ki67 (Abcam, cat no. ab15580, Cambridge). The sections were heat treated for antigen retrieval by autoclaving at 121 °C for 15 minutes in 0,01M citric acid pH 6.0 for CD3 and Ki67, and in the microwave in pH 6.6 Target Retrieval Solution (DAKO, Glostrup, Denmark) for CD18 and pH 9.1 tris-EDTA buffer for β-catenin. Page 7/17 Endogenous peroxidase activity was inhibited with blocking reagent for 10 minutes (DAKO Envision system-HRP AEC REF K 4009 for CD3 and 3.0% H2O2 in methanol for Ki67, β-catenin and CD18). Non- Page 7/17 Page 7/17 specific antigenic sites were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) for CD3, 1% normal goat serum (Vector/Bioteam) in 5% BSA/TBS for Ki67, 2% BSA in TBS for β-catenin and 10% normal goat serum in PBS for CD18. The sections were incubated at room temperature with the following primary antibodies, dilutions and incubation times: rabbit anti-CD3 (60 minutes), rabbit anti- Ki67 (1:1000 in 2,5% BSA/ TBS, 60 minutes), mouse anti-dog-CD18 (1:100 in 10% goat serum, 30 minutes) or mouse anti-ß-catenin (1:2500 in 1% BSA/TBS, 60 minutes). Sections were then incubated for 30 minutes with secondary antibody from the DAKO Envision-kit for CD3, CD18 and ß-catenin, and goat anti rabbit (DAKO, E 432) diluted 1:50 with 2% normal goat serum for Ki67. The Ki67- sections were then incubated for 30 minutes with Elite -ABC- kit (VECTASTAIN PK-6100) at diluted 1:50 in TBS. Colour was revealed for 10 to 15 minutes using DAKO Envision system-HRP AEC for CD3, CD18 and ß-catenin, and the substrate solution (IMMPACT AEC PEROXIDASE SUBSTRATE SK-4205) for Ki67. Between the various steps, the sections were rinsed thoroughly in TBS. Finally, the sections were counterstained with haematoxylin solution for 45 seconds and mounted. Negative control staining was performed by replacing the primary antibodies with non-immunized goat serum, and showed no staining. Evaluation Of Immunohistochemistry The sections were blinded and analyzed subjectively. Two pathologists evaluated the IHC score individually and agreed on the final score (CD18 and β-catenin; GG and RR, Ki67 and CD3; GG and ØK). For all sections, the IHC score was determined by evaluating the entire specimen. For β-catenin, the scoring scheme included prevalence of cells with a positive staining nucleus, using the following grading system: 0: no cells have any positive staining nucleus, 1: <1/3 of the cells have positive staining nucleus, 2: 1/3–2/3 have positive staining nucleus and 3: > 2/3 of the cells have a positive staining nucleus. Furthermore, the intensity of the β-catenin staining in the cytoplasm and the nucleus were scored from 0–3 (no staining, weak staining, moderate staining and strong staining). The Ki67 staining was considered as nuclear staining in cells, and absence of nuclear staining was considered negative for the antigen. The scoring of Ki67 was only evaluated in the intraepithelial compartment. None of the control samples expressed Ki67, thus the IHC scoring of Ki67 were only reported in tumour samples. CD3 + cells were defined as clearly stained cytoplasm in the epithelial compartment and within cells in the lamina propria. CD3 + cells were defined as clearly stained cytoplasm in the epithelial compartment and within cells in the lamina propria. For CD3, Ki67 and CD18, a semi-quantitative scoring scheme based on the prevalence of positive staining cells using the following grading system was applied: 0: no staining, 1: few positive cells, 2: a moderate amount of positive cells, and 3: many positive cells throughout the examined tissue. The scores of the two pathologists were averaged, resulting in one score for each variable. If the difference between the two pathologists deviated by more than one grade (8 out of 228 scores), the slides Page 8/17 were reviewed and discussed, resulting in a final score. Statistical analysis The IHC score between adenoma and adenocarcinoma were compared, but due to the low number of samples diagnosed as adenocarcinomas, the adenoma and adenocarcinoma were categorized as “tumour samples” when comparing the IHC score to controls. The difference in demographic factors and the IHC score between dogs with adenoma and adenocarcinoma (tumour samples) and control dogs were analysed using non-parametric tests (Wilcoxon test) JMP 14 (SAS, USA). A P-value < 0.05 was considered significant for all statistical tests. Results No significant difference in breed, age and gender were noted among dogs with colorectal adenoma, adenocarcinoma and control dogs (Wilcoxon test, p > 0,1). The following breeds contributed with ≥ 3 samples: German Shepherd (n = 4), English Setter (n = 3) and Cocker Spaniel (n = 3). The control dogs consisted of various breeds, of which none contributed with more than one sample in the study material. Only five out of 23 tumour samples were diagnosed as adenocarcinomas; the remaining were adenomas. Only five out of 23 tumour samples were diagnosed as adenocarcinomas; the remaining were adenomas. Only five out of 23 tumour samples were diagnosed as adenocarcinomas; The tumours were located in rectum in 15 dogs, in colon in four, while the localization of the remaining four tumours were not specified (Table 1). The tumours were located in rectum in 15 dogs, in colon in four, while the localization of the remaining four tumours were not specified (Table 1). The number of CD3 + cells in the epithelial compartment was significantly lower in tumour samples compared to control samples (Wilcoxon test, p = 0,0006). No difference in the lamina propria CD3 + cell- number were detected between tumour and control samples (Wilcoxon test, p > 0,1), (Fig. 1A and B). The number of CD3 + cells in the epithelial compartment was significantly lower in tumour samples compared to control samples (Wilcoxon test, p = 0,0006). No difference in the lamina propria CD3 + cell- number were detected between tumour and control samples (Wilcoxon test, p > 0,1), (Fig. 1A and B). The tumour samples had lower expression of CD18 positive cells in the lamina propria, compared to control samples (Wilcoxon test, p = 0,001) (Fig. 1C and D). The tumour samples had lower expression of CD18 positive cells in the lamina propria, compared to control samples (Wilcoxon test, p = 0,001) (Fig. 1C and D). The Ki67 positive cells showed a strong signal in adenomas and adenocarcinomas, but no significant difference in expression levels were detected between these two tumour stages (Wilcoxon p > 0,05) (Fig. 1E and F). The Ki67 positive cells showed a strong signal in adenomas and adenocarcinomas, but no significant difference in expression levels were detected between these two tumour stages (Wilcoxon p > 0,05) (Fig. 1E and F). Discussion Colorectal carcinoma develops from clonal expansion of genetically altered cells in humans (9) and in dogs (10–14). This process is also believed to be influenced by inflammation (26). Inflammatory cells may reduce the risk of metastasis and further improve the prognosis (21, 23). Low infiltrations of T-cells evaluated by CD3 in CRC was associated with reduced survival in humans (23, 24). In our work, epithelial CD3 + cells were reduced in colorectal adenomas and adenocarcinomas compared to healthy colonic tissue. Moreover, the amount of CD3 cell infiltration was not different in adenomas compared to adenocarcinomas. This may indicate that T-cells is not a major player in the colorectal carcinogenesis of dogs, or that a reduced number of T-cells may cause a reduced immune defence against tumour progression. However, an expression of different subpopulations of T-cells amongst tumour and controls samples and any significance of this, cannot be excluded.T-cells are divided in subpopulations depending on their functions (27, 28). CD8 + and CD4 + T-cells serve different functions, with cytotoxic and immunomodulating properties, respectively. In humans, the T-cells in the epithelial compartment consist mostly of CD8 + cells, whereas the CD4 + cells dominate the lamina propria, both in normal colonic mucosa and in CRC stroma (29). However, in the lamina propria of the colon, the number of CD4 + cells were lower than the CD8 + cells (30). In the healthy small intestine of dogs, more CD8 + cells are present in the epithelial compartment compared to the lamina propria, where the CD4 + cells predominate. Studies of dogs with inflammatory bowel disease have observed higher infiltrations of CD4 + cells in lamina propria in duodenal mucosa compared to control dogs (31). Another study of dogs with steroid- responsive diarrhea and food-responsive diarrhea that characterized CD4 + cells in duodenal and colonic mucosa observed a lower number of these cells. Furthermore, treatment with corticosteroids or hydrolyzed diet, did not change the magnitude of CD4 + cell infiltration (32). These studies indicate a lower presence of CD4 + cells in the lamina propria in colon compared with the duodenum, and that inflammation is present despite clinical response to treatment. Thus, identifying mucosal inflammation correlating with the clinical signs is challenging. FoxP3 is a marker for T-regulatory cells, and the presence of these cells may inhibit an efficient immune defense against tumour development (33). Results Colonic samples from control dogs had a uniform staining of β-catenin along the cell membrane of epithelial cells. When compared to normal colonic epithelial cells, the expression levels of cytoplasmic β- catenin were significantly higher in adenomas and adenocarcinomas (Wilcoxon test, p = 0,0002). (Fig. 1G and H). Colonic samples from control dogs had a uniform staining of β-catenin along the cell membrane of epithelial cells. When compared to normal colonic epithelial cells, the expression levels of cytoplasmic β- catenin were significantly higher in adenomas and adenocarcinomas (Wilcoxon test, p = 0,0002). (Fig. 1G and H). None of the control samples showed any positive staining of β-catenin in the nucleus of colonic cells. In contrast, 14/23 of the tumour samples had moderate or strong staining of the nucleus. This difference in Page 9/17 β-catenin expression (signal strength and distribution) in nucleus of colonic cells between controls and tumours, was statistically significant (Wilcoxon test, p = 0,0002). No significant difference was detected for nuclear β-catenin expression (signal strength and distribution) between adenoma and adenocarcinoma (Wilcoxon test, p > 0,05) Results of the IHC-scores are found in Additional file 1. Discussion Studies in humans with colorectal cancer have found an association between a low CD3+/FoxP3 ratio and shortened survival (34). Future studies should also aim to characterize the T-cell subpopulations including FoxP3, CD4 + and CD8 + cells in canine colorectal adenoma and adenocarcinomas. Page 10/17 In humans, inflammation dominated by lymphocytes and plasma cells is higher in CRC compared to normal colonic mucosa (29) and non-steroidal anti-inflammatory treatment seems to be effective (35). Despite our results with a reduced presence of T-cells and histiocytes in canine colorectal tumours, anti- inflammatory treatment is often effective. Dogs with rectal polyps of various malignancy, with minor infiltration of inflammatory cells treated with piroxicam rectally, improved with regards to clinical signs and size of tumours, indicating that suppressing inflammation in these cases may be advantageous (36). Results from our study confirmed what is previously described, that β-catenin may be useful as a marker for tumour progression, as its expression is increased in cytoplasm and nucleus of cancerous cells compared to healthy colon (10, 16, 37). A study showed that the protein may correlate with the tumour stage (10). However, in the present study, the magnitude of nuclear location of β-catenin did not appear to correlate with the tumour stage, as our adenocarcinoma samples were not demonstrating a stronger staining of nuclear β-catenin than the adenomas. This has also earlier been observed(16). However, subtle changes may not be detected only by using light microscope. Other methods such as electron microscopy may be more sensitive for this purpose. Anyhow, the lack of consistency among studies may be explained by several factors, like other criteria for tumour classification, including World Health Organization classification of tumours in domestic animals (38). The dogs with colorectal adenoma and adenocarcinoma consisted of various breeds, including German Shepherd and Cocker Spaniel, each represented by three individuals. These breeds are previously described as dominant in case-reports of dogs with colorectal adenoma and adenocarcinoma (8, 36, 39, 40). In the majority of dogs (15/19 dogs), tumours were present in the rectum, and only four dogs had tumours in the colon. Similarly, in a study of 78 dogs with colorectal adenocarcinomas, over 85% of dogs had rectal tumours (6). Acknowledgements The authors would like to thank the veterinary students Iselin Lyngholm Klinkenberg and Jorunn Karina Skadsem Gil who contributed with the preparation of samples for immunohistochemistry analyses. Discussion As we used strict criteria for classifying a tumour as an adenocarcinoma, most tumours were diagnosed as adenomas and the low number of adenocarcinomas was a limitation of this study Thus, comparing the IHC score between adenomas and adenocarcinoma was challenging. Moreover, limitations by using archived samples include inadequate information of clinical data. The control dogs represented a heterogeneous group, but were within similar age-groups as the dogs with tumours. Commonly, control dogs are healthy young laboratory dogs (16). As age affects the degree of gastrointestinal inflammation (41, 42), it may be more relevant to include older dogs as controls, as was done here. The need for markers to distinguish adenomas and adenocarcinomas, and thus evaluate prognosis is necessary. In humans, identifying the density of CD3 + and CD8 + T-cells in the core and margin of tumours may aid this purpose (43). This methodology named “immunoscore” may also be useful to separate adenomas from adenocarcinomas in dogs, and future studies should aim to determine the types and magnitudes of immune cells infiltrating canine colorectal tumours. Page 11/17 Page 11/17 The lower presence of CD18 and CD3 + cells in tumours compared to controls in this study indicates a reduced presence of T-cells, which may be of importance in future investigation and understanding of development and immunophenotype of canine colorectal cancer. The lower presence of CD18 and CD3 + cells in tumours compared to controls in this study indicates a reduced presence of T-cells, which may be of importance in future investigation and understanding of development and immunophenotype of canine colorectal cancer. Funding The Norwegian Research Foundation for Canine Cancer provided financial support. The Norwegian Research Foundation for Canine Cancer provided financial Availability of data and materials All data generated or analysed during this study are included in this published article and its additional files. Authors' contributions KH, ES and GG designed the study. LT and RR performed laboratory work. ØK, GG and RR performed the IHC scoring. KH performed statistical analysis. KH wrote the manuscript with contributions from all authors during manuscript preparation. All authors read and approved the final manuscript. Competing interests The authors declare no conflict of interest. Ethics approval and consent to participate Owner consent for using leftover samples for research in cancer diseases was given by dog-owners. These samples had been taken for clinical purposes and leftover samples were archived and used for this study. Consent for publication Not applicable References 2. Lingeman CH, Garner FM. Comparative study of intestinal adenocarcinomas of animals and man. 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A small number of positive cells were found in the lamina propria (arrow). 40x. B. CD3, normal colon. Scattered cells and clusters of cells were seen in the lamina propria (arrow) and the epithelium. 40x. C. CD18, adenocarcinoma. Fewer positive cells (arrow) and weaker signal than in the control. 10x. D. CD18, control. Characteristic distribution of positive cells, scattered in the lamina propria () and concentrated below the crypt epithelium (arrows). 10x. E. Ki-67, Figure 1 Page 16/17 Immunohistochemistry. A. CD3, adenocarcinoma. A small number of positive cells were found in the lamina propria (arrow). 40x. B. CD3, normal colon. Scattered cells and clusters of cells were seen in the lamina propria (arrow) and the epithelium. 40x. C. CD18, adenocarcinoma. Fewer positive cells (arrow) and weaker signal than in the control. 10x. D. CD18, control. Characteristic distribution of positive cells, scattered in the lamina propria () and concentrated below the crypt epithelium (arrows). 10x. E. Ki-67, Page 16/17 Immunohistochemistry. A. CD3, adenocarcinoma. A small number of positive cells were found in the lamina propria (arrow). 40x. B. CD3, normal colon. Scattered cells and clusters of cells were seen in the lamina propria (arrow) and the epithelium. 40x. C. CD18, adenocarcinoma. Fewer positive cells (arrow) and weaker signal than in the control. 10x. D. CD18, control. Characteristic distribution of positive cells, scattered in the lamina propria () and concentrated below the crypt epithelium (arrows). 10x. E. Ki-67, Page 16/17 Page 16/17 adenocarcinoma. Numerous cells have strong, nuclear staining (arrows). 40x. F. Ki67, adenoma. Numerous cells have strong, nuclear staining (arrows). 40x. G. β-catenin, adenocarcinoma. Strong staining of neoplastic epithelial cells. Both cytoplasmic (arrows) and nuclear (arrowheads) staining are evident. 40x. H. β-catenin, control. Weak staining of epithelial cell cytoplasm only (arrow). 40x. adenocarcinoma. Numerous cells have strong, nuclear staining (arrows). 40x. F. Ki67, adenoma. Numerous cells have strong, nuclear staining (arrows). 40x. G. β-catenin, adenocarcinoma. Strong staining of neoplastic epithelial cells. Both cytoplasmic (arrows) and nuclear (arrowheads) staining are evident. 40x. H. β-catenin, control. Weak staining of epithelial cell cytoplasm only (arrow). 40x. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. Manuscriptreferences.pdf Additionalfile1.docx Page 17/17
https://openalex.org/W2145055477	https://geoenvironmental-disasters.springeropen.com/counter/pdf/10.1186/s40677-014-0008-z	English	null	The geotechnical characteristics of landslides on the sedimentary and metamorphic terrains of South-East Nigeria, West Africa	Geoenvironmental disasters	2,015	cc-by	7,101	Abstract Background: Landslides in Nigeria occur in various forms and vary in mode, scale and frequency. The variations appear to be significantly controlled by geologic setting, hence the need to study in detail the distinctive features that differentiate landslides in sedimentary environments from those on metamorphic localities. The aim is to understand the actual features impacted on the landslides by geology, from which future predictions of occurrence and identification of instability could be based. The recognition of features that are characteristic of certain geologic setting may be a major step in early warning development in Nigeria. To achieve the objectives detailed mapping of the study area was carried out using topographic maps, aerial photographs and multiple field surveys. Results: The landslides on the sedimentary terrain were mainly shallow, low-volume movements, material slumps and short runout slides some of which activated on slopes that followed the dip of strata, along wavy shear surfaces controlled by impermeable bedding planes. Runoff-triggered movements caused by erosion of channel bed and banks and failures caused by sediment bulking of runoff with material eroded from headwater slopes are also common on the sedimentary terrain. Conclusions: In comparison, the landslides on the metamorphic terrain were complex translational and rotational movements and mudslides on steep slopes sometimes involving a combination of slide and flow with curved headscarps and slickensided shear surfaces. The looseness of slope materials and their relatively low strength parameters account for the dominance of landslides on the sedimentary zone. Keywords: Landslides; Sedimentary rocks; Metamorphic terrain; Strength parameters; Stability Keywords: Landslides; Sedimentary rocks; Metamorphic terrain; Strength parameters; Stability result in damage and fatalities (Petrucci and Polemio 2009). In West Africa, landslides are caused primarily by rainfall. Depending on meteorological and geomorpholo- gic conditions, individual rainfall events can trigger small or large slope failures. Liao et al. (2012) defined the rela- tionship between factor of safety and rainfall on an infinite slope by means of a physical model known as SLope- Infiltration-Distributed Equilibrium (SLIDE) which took into account the effect of water infiltration on the stability of slopes. Landslides induced by high-intensity or pro- longed rainfalls constitute a major risk factor in Nigeria especially because they have generally been poorly defined in the past. The landslides have the potential to damage hu- man settlements, industrial development, cattle ranch, for- estry, and agricultural activities. For instance in October 2008 at 3:30 a.m. Abstract local time, a mudflow occurred 200 km east of Enugu metropolis, south-east Nigeria. During a rain © 2015 Igwe; licensee Springer. 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 credited. Igwe Geoenvironmental Disasters (2015) 2:1 DOI 10.1186/s40677-014-0008-z Open Access Open Access The geotechnical characteristics of landslides on the sedimentary and metamorphic terrains of South-East Nigeria, West Africa Ogbonnaya Igwe Background Landslides are a major hazard in Africa where resources worth several millions of dollars are lost annually during seasons of heavy and also light rains. The mechanisms of rainfall-induced landslides have been extensively stud- ied and some of the conclusions assert that the amount of rain, nature of slope-material, discontinuities and weathering are the major factors predisposing a slope to failure (Iverson 2000; Msilimba and Holmes 2010; Wang et al. 2002; Sassa et al. 2004; Guzzetti et al. 2008). Water- infiltration is a significant triggering factor for slope fail- ures around the world. Prolonged and/or intense rainfall can trigger landslides, floods and secondary floods (stag- nancy of rain on low permeability surfaces) that could Correspondence: igwejames@hotmail.com Department of Geology, University of Nigeria, Nsukka, Nigeria Correspondence: igwejames@hotmail.com Department of Geology, University of Nigeria, Nsukka, Nigeria Igwe Geoenvironmental Disasters (2015) 2:1 Page 2 of 14 storm, a portion of an unstable hill (< 500 m) collapsed, sending mud and debris across the rural neighborhood kill- ing 2, damaging agricultural farms and covering an area of approximately 1.3 km2. Recognition, identification and mechanisms of such landslides has received intense study (Wei et al. 1993; Sassa 1998; Aleotti 2004; Wang et al. 2005, 2009; Sassa et al. 2004; Wang and Sassa 2010), how- ever a few studies have attempted a systemic evaluation and grouping of landslides according to their geologic ori- gin. While landslide susceptibility maps attempt to delin- eate areas with potential for future failures, they appear silent on the distinctive features locality can impact on slope movements. A comprehensive landslide risk assess- ment should therefore not only include investigation of landslide processes and formulation of procedures for haz- ard reduction, but also the identification of imprints of geologic setting on the shape, size (morphology) and mo- bility of landslide. To understand morphology and mobil- ity, detailed analysis of the source, location, severity, recurrence interval, triggering and displacement mecha- nisms are important areas of consideration. Figure 2 The International tourist area of Obudu, Calabar (photo by Igwe). Figure 2 The International tourist area of Obudu, Calabar (photo by Igwe). and steepness of slopes in the area. These differences in scale and frequency were the major motivating factors for the research. Although the landslides vary in scale and frequency in the two areas, they are all shallow, rainfall-induced landslides. Background Wieczorek and Glade (2005) and Gabet and Mudd (2006) reported that debris flows can be mobilized from shallow landslides. The landslide debris frequently creates inconveniences by destroying communication and hydraulic facilities, or by blocking roads and communication routes. Sometimes death also Two localities (Calabar and Enugu, Figure 1) that dif- fer in geology and land use were studied. While land- slides are common and frequent in the hilly parts of Enugu because of its unique geology, long-time residents report that the recent landslides at the international tourist town of Obudu in Calabar (Figure 2) are the first known slope failures despite the much higher elevation Figure 1 Location of the study areas. Figure 1 Location of the study areas. Figure 1 Location of the study areas. Page 3 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Figure 5 Some of the casualties of the Obudu landslide were the occupants of a car that tumbled when a slope near the road failed (photo by Igwe). Figure 3 Shallow slides at the sedimentary terrain in Enugu (photo by Igwe). Figure 3 Shallow slides at the sedimentary terrain in Enugu (photo by Igwe). Figure 5 Some of the casualties of the Obudu landslide were the occupants of a car that tumbled when a slope near the road failed (photo by Igwe). occurs when displaced landslide masses crash on build- ings or into people. The prolonged rainfall of October 2013 triggered 28 new shallow landslides in Enugu (some of which are shown in Figure 3) and four pro- nounced landslides at Obudu area of Calabar (one of such is shown in Figure 4). Some of the landslides brought untold hardship to international tourist who had flocked to Obudu to catch a glimpse of the beauty of the fading sun on the Obudu Hills during the months that de- marcate the wet and dry seasons. These landslide events caused casualties (one such casualty from the ditched ve- hicle in Figure 5) and severe economic losses and illus- trated the need for the risk assessment through proper understanding of the geotechnical and hydrological char- acteristics of the slopes. Knowledge of the mechanisms of precipitation-induced slope failures is of great importance in the management of landslide hazards, especially be- cause there is no systematic monitoring of slopes in Nigeria. Marchi et al. (2002) reported the usefulness of slope monitoring in hazard and risk assessment. Background The objective of this study is two-fold: (1) to carry out a medium to large-scale assessment of landslides in two vulnerable localities in south-east Nigeria that lie on two different geology – sedimentary and metamorphic, (2) to understand their geotechnical properties and attempt to develop a size-predictive model that may contribute to fu- ture preventive and management measures. General procedure Detailed mapping of the study area was carried out using topographic maps in the scale of 1: 50,000, aerial photo- graphs and multiple field surveys, The major goal of the study was to identify areas frequently affected by land- slide episodes and compare their hydro-geological and geotechnical characteristics. By comparing these charac- teristics in areas prone to the hazard, susceptible zones and initiation mechanisms could be properly under- stood. Several documents that provided historical data on past landslides were reviewed and chronologically sorted to determine cases that had been the most dam- aging to lives and property. The landslide types were classified in accordance with Cruden and Varnes (1996). Landslide locations in the study area were identified from interpretations of aerial photographs and field sur- veys. During the mapping, aerial photographs (taken in the 1970s and 80s) and topographic maps at a scale of 1:50,000 were evaluated. Validation of the aerial photos was later done by carrying out multiple field surveys of the study site. Measurements essential to the preparation of an adequate geologic map were taken at various loca- tions within the study area. Such study has not been car- ried out on the Nigerian landscape. Figure 4 A landslide on the metamorphic terrain in Obudu Calabar (photo by Igwe). Figure 4 A landslide on the metamorphic terrain in Obudu Calabar (photo by Igwe). Figure 4 A landslide on the metamorphic terrain in Obudu Calabar (photo by Igwe). Page 4 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Landslide bodies were mapped from crown to toe of rupture. Similar method was reported by Aleotti and Chowdhury (1999). Note was taken of the slope height, slope length, slope gradient, lithology, and dip direction of slope, the thickness and slope of the sliding surface. Original geometry of slope, position of water table and shear strength parameters were also analyzed. Depths were estimated from trial pits and boreholes. Geological cross-sections were then made to study the morphology of the slides. The soils’ cohesion intercept (c) and angle of shearing resistance (ϕ) were comprehensively determined with a series of consolidated, undrained, and saturated tri- axial tests. Using the geological and geotechnical data, in- terpretations were made and conclusions drawn. Precipitation record was obtained from metrological stations near the landslides. General procedure Data obtained included the mean annual precipitation, the average number of rainy days, the precise or approximate location of the area af- fected by the landslides, the precise or approximate time, date, or period of the failures, the rainfall conditions that resulted in slope failures and the number of landslides triggered by rain. Such information has not been reliably or sufficiently documented. Analysis was then made of the geographical distribution of the rainfall and landslide events. An apparent limitation in the use of the rainfall information was the unavailability of many rain gauges in the study areas. This prevented decisions on the reli- ability of a rain gauge based on the geographical distance Figure 6 Local Geology of Enugu study site with cross sections. Figure 6 Local Geology of Enugu study site with cross sections. Figure 6 Local Geology of Enugu study site with cross sections. Page 5 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 to the Eocene in age and were deposited in environments that pass from continental in the proximal parts through paralic to shallow marine in the distal parts (Kogbe, 1989; Oboh et al. 2005). The oldest deposits with the Anambra basin belong to the Campanian Nkporo Group composed of the diachronous Enugu Shale formation, Nkporo shale formation and the Owelli Sandstone formation. Rocks of the Mamu Formation outcrop in the study area and con- sist of bluish dark grey shales containing abundant car- bonaceous matter with thin siltsone and sandstone layers. The Mamu Formation was deposited after the Enugu to the landslide (or landslide area), the elevation of the rain gauge compared to the elevation of the landslide, and the location of a rain gauge with respect to the local topographical and morphological setting. Generally, the study used the information from any available rain gauge closest to the landslide area. Site characteristics Geology The dominant rock types in Enugu area are sandstone silt- stone and shale (Figure 6) that range from the Campanian Figure 7 Geology at the metamorphic locality. Figure 7 Geology at the metamorphic locality. Page 6 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Table 1 Select geotechnical characteristics of the slope materials Table 1 Select geotechnical characteristics of the slope materials Specimen No. Terrain type Average frictional resistance (o) Average cohesion (kPa) Average coefficient of permeability (cm/sec) Enugu Group 1 sedimentary 25 11 3 × 10−2 Enugu Group 2 sedimentary 13 10 1 × 10−4 Enugu Group 3 sedimentary 15 10 1 × 10−3 Enugu Group 4 sedimentary 25 16 2 × 10−4 Calabar Group 1 Metamorphic 18 20 4 × 10−4 Calabar Group 2 Metamorphic 16 25 3 × 10−4 Calabar Group 3 Metamorphic 18 25 1 × 10−5 Calabar Group 4 Metamorphic 18 40 2 × 10−5 Calabar Group 5 Metamorphic 18 40 3 × 10−4 Intense and short duration rainfalls characterize the outset of rainy season in the area. Annual rainfall (based on 2012 precipitation amount) ranges between 1500 mm to 2100 mm (Igwe et al. 2013). The areas lie between the tropical rainforests which dominate nearly half of southern Nigeria and is characterized by luxuriant vegetation and abundant plant species. It is bounded by fresh water swamp forest in the south and Guinea Savanna in the North. The vegetation is marked by continuous growth of trees, shrubs and climbing plants. shale and consists of heteroliths of siltstone, shale and fine grained sands with coal seams. The Maastrichian Ajali Sandstone Formation lies conformably on the Mamu For- mation. It consists of characteristically friable, cross- bedded sandstones with a virtual absence of clay. The Ajali sandstone which is about 406 m thick in the area is over- lain by lateritic/red earth deposits. The dominant rock types in Obudu, Calabar are gneisses, migmatites, amphibolites, quartzites, schists, granites and pegmatites (Ekwueme 1991). According to Ukaegbu and Oti (2004) and Ukaegbu and Beka (2007) the basement rocks of the Obudu plateau have undergone amphibolite to granulite facies metamorphism and consist dominantly of the aforementioned rocks (Figure 7). Additionally, poly- phase deformation has caused folding, refolding, faulting, foliation and shearing of the metamorphic rocks with mainly N-S to NE-SW trends in conformity with trends in other parts of the reactivated Basement Complex of Nigeria. The Obudu tourist area landslide This research is the first work distinguishing landslide features on two different terrains in Nigeria. Obudu hills Figure 8 Some of the landslides in Obudu cut power supply by damaging or deforming the electric lines (photo by Igwe). For geotechnical investigations, the entire road stretch was divided into 16 uniform slope sections based on slope angle and rock types following the method es- poused in Dai et al. (2002), Chen and Wang (2007) and Das et al. (2010). Site characteristics Geology They reported that the tectonic imprints are reflected as remnant Pre-Pan-African (E-W to NW-SE) to dominant Pan-African (N-S to NE-SW) structural features. The area is considered the terminal end of the western prolongation of the Cameroon Massif (Edet et al. 1994; Ejimofor et al. 1996; Ekwueme, 1998; Toteu et al. 2004). Due to the movement of meteorological perturbations and the heat/moist exchange with the sea the rainiest areas are the southern parts. Nevertheless, the northern parts are sometimes hit by very intense storms because of enhancement due to the relief. During such periods, daily rainfall could increase to 20–30% of mean annual rainfall in the area. Climatic features Calabar and Enugu have similar tropical climate, with dry summers (monthly minimum or zero rainfall in from November to March) and wet season (widespread monthly high rainfall from April to October). The mean annual temperature at Obudu varies from 14°C to 28°C and mean annual rainfall of approximately 2000 mm to 3000 mm (Ekwueme 1991; Ejimofor et al. 1996). The mean monthly temperatures in Enugu vary from 22°C to 28°C in the wet season and between 28°C and 32°C in the dry season. Figure 8 Some of the landslides in Obudu cut power supply by damaging or deforming the electric lines (photo by Igwe). Page 7 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 are located in the Obanliku Local Government Area of Cross River State, southeastern Nigeria. They lie be- tween latitudes 6°20’ and 6°30’ N and longitudes 9°20’ and 9°30’ E (Figure 7). The area is about 104 m2 and stand at a height of about 1576 m above sea level (Ekwueme, 2003). The geotechnical properties of the slope materials are summarized in Table 1. slope materials, make assessment of water table depths and ground-water flow patterns difficult. To validate the results from the hand-dug wells, geophysical method – vertical electrical sounding – was utilized. The results agree with the observation wells’ data. Field observations showed that the landslides on the metamorphic terrain have discrete, slickensided shear surfaces on which analyses were possible. This was a major difference between mass movements on the meta- morphic and sedimentary landscape. There were rupture surfaces from which movements began as slides and continued for long distances as mudflow/slides. The ma- terials involved were typically poorly-sorted sandy collu- viums supported by about 43% clay matrix produced On October 14th 2013, after many days of heavy rain with cumulative precipitation of over 600 mm, several debris slides were triggered in the mountain of Obudu. Some of the landslides blocked the only access route to the sightseeing area, while others cut power supply (Figure 8). The water table varied from 0.9 to 4 m. The undulating topography, and non-homogeneous or anisotropy of the Figure 9 The landslide debris initially covered the road before being cleared in (A) and damaged power cables prompting the erection of a new power line in (B) (photo by Igwe). Climatic features Figure 9 The landslide debris initially covered the road before being cleared in (A) and damaged power cables prompting the erection of a new power line in (B) (photo by Igwe). Page 8 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 from the weathering of the rocks. The consistency of such materials is usually closer to the plastic rather than the liquid limit (Hutchinson 1988). The characteristics the clay matrix probably contributed to the rapid move- ment witnessed on the metamorphic terrain. For long distance travel, motion down the slope may be facilitated by undrained loading (Hutchinson and Bhandari 1971; Wang et al. 2000, 2002). One of the landslides involved mass movement of 19,000 m3 materials downslope for 7 m (from the head scarp to the toe) covering the sec- tion of the road and completely blocked access to the hills or the city centre and destroyed some power cables (Figure 9A, B). The thickness of the regolith varied from 0.2 m to 0.7 m in some places. The slope elevation and inclination were 790 m and 62° respectively. The access road was promptly cleared of debris by the Municipal government authorities to restore confidence in the for- eign visitors. Direct field observation and photographs taken appear to show the presence of discontinuities and lines of weakness. Field observation and slope stability analysis revealed that sliding of the top block (failed block, Figure 9B) was likely. Dip slope failure of the regolith directly above the fine materials is possible. therefore not a coincidence that all the dangerous land- slides occurred along the roads axes. Excluding gravelly materials in the failed masses, the average sand, silt and clay content of the samples is 52%, 34% and 14% re- spectively. The average range of coefficient of perme- ability was 4 × 10−4 – 2 × 10−5 cm/sec. At one of the exposed sections of a landslide on the slope, three layers of varying thickness and permeability were observed (Figure 9A). The calculated average vertical permeability kv of the three-layered profile visible at the landslide site was about 0.84 × 10–3 cm/sec. While the average range of the angle of internal resistance and cohesion were (16° – 18°) and (25 kPa – 40 kPa) respectively. The Enugu landslides Slumps and short runout debris slides of limited volume are frequent and many at this location in spite of the areas relatively low elevation and slope angle. The slow to rapid of the mostly unsaturated movements usually ini- tiate within the sandy body or on clayey/shale interlayer, but are typically on non-discrete, matrix-poor shear sur- faces. The movements were mainly non-confined and without established channel. The slope material is domin- antly unconsolidated, friable sands that easily give way during intense rain. The average content of sand, silt and clay is 75% sand, 16% silt and 9% clay with uniformity co- efficient of 2.8. The average range of coefficient of perme- ability was 3 × 10−2 – 2 × 10−4 cm/sec. The slope is dominantly a two-layered profile with a calculated verti- cal permeability, kv of 1.6 × 10−2 cm/sec. The average range of the angle of internal resistance and cohesion were (13° – 25°) and (10 kPa – 16 kPa) respectively. The recent landslides in the area are the first known landslides in the hills, indicating high stability. There had been no previous landslide experience on the slopes al- though the slopes are quite steep and stand at high eleva- tions. The area is hilly with some peaks reaching 1000 m. Access roads (which obviously decreased stability) were constructed to improve scenery and attract tourist. But during or after the road construction, no slope stabilization technique was put in place to check sliding. It is Figure 10 Enugu landslide and its lithologic description (photo by Igwe). Figure 10 Enugu landslide and its lithologic description (photo by Igwe). The Enugu landslides Page 9 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Table 2 Summary of field measurements from the sedimentary terrain Longitude (E) Latitude (N) Length (m) Width (m) Slope angle (o) Elevation (m) 007 °26'13.4" 06°28'40.6" 6 3.2 20 309 007 °26'22.3" 06°28'57.7" 9.4 3.5 14 307 007 °26'12.2" 06°28'40.3" 6.08 2.9 18 314 007 °26'12.5" 06°28'40.1" 4.6 2.8 16 315 007 °26'12.0" 06°28'40.8" 4 4.5 31 311 007 °26'10.0" 06°28'46.1" 15 7.73 32 337 007 °26'09.0" 06°28'45.6" 8 6 10 349 007 °26'08.7" 06°28'46.0" 12 8 29 353 007 °26'09.1" 06°28'46.1" 8 5 32 350 007 °26'10.1" 06°28'46.4" 20 10 20 354 007 °26'09.0" 06°28'46.6" 15 9 55 358 007 °26'08.8" 06°28'46.7" 13 8 40 354 007 °26'05.9" 06°28'48.8" 18 12 35 368 007 °26'06.1" 06°28'49.0" 10 5 32 370 007 °26'06.3" 06°28'49.0" 9 5 45 370 007 °26'06.4" 06°28'48.8" 6 3 35 366 007 °26'06.2" 06°28'48.6" 10 4 33 364 007 °26'06.4" 06°28'48.6" 6 3 34 365 007 °26'06.4" 06°28'48.6" 1.5 0.5 35 368 007 °26'06.8" 06°28'49.3" 25 10 40 375 007 °26'06.9" 06°28'48.6" 9 4 30 366 007 °26'07.5" 06°28'49.4" 15 9 40 374 007 °26'07.9" 06°28'48.7" 7 3 60 363 007 °26'08.2" 06°28'48.6" 8 4 35 364 007 °26'08.2" 06°28'48.9" 6 4 30 363 007 °26'08.4" 06°28'48.8" 2 0.5 32 359 007 °26'08.6" 06°28'48.9" 4 0.5 30 357 007 °26'08.6" 06°28'48.7" 5 3 30 353 007 °26'08.6" 06°28'49.1" 20 10 28 359 007 °26'08.6" 06°28'49.3" 3 1 46 361 007 °26'09.2" 06°28'48.9" 1 0.7 20 351 007 °26'09.7" 06°28'49.1" 12 4 40 357 007 °26'09.0" 06°28'49.4" 1.5 0.7 35 351 007 °26'08.9" 06°28'49.3" 2 0.5 30 352 007 °26'08.8" 06°28'49.3" 1 0.25 28 353 007 °26'08.7" 06°28'49.6" 5 3 33 359 007 °26'08.5" 06°28'49.7" 5 2.5 35 361 007 °26'08.2" 06°28'49.6" 2.5 1 32 363 007 °26'08.2" 06°28'49.4" 3 1 35 365 007 °26'08.1" 06°28'50.4" 12 4 31 377 007 °26'08.4" 06°28'50.8" 9 5 24 375 007 °26'08.7" 06°28'50.7" 5 6 28 373 007 °26'08.9" 06°28'50.4" 5 3 22 371 007 °26'09.0" 06°28'50.3" 2.5 2 25 370 Table 2 Summary of field measurements from the sedimentary terrain Page 10 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Table 2 Summary of field measurements from the sedimentary terrain (Continued) 007 °26'08.9" 06°28'50.1" 7 2.5 29 367 2 007 °26'08.6" 06°28'49.1" 1.5 0.5 60 352 1 007 °26'03.4" 06°28'59.4" 7 3 20 404 1 007 °26'03.3" 06°29'01.1" 3 1.5 30 406 2 007 °26'03.3" 06°29'01.3" 5 2.5 35 407 1.5 007 °26'02.3" 06°28'57.4" 6 2.5 35 394 1 007 °26'02.6" 06°28'55.9" 3 1.5 25 388 0.5 007 °26'04.7" 06°28'50.6" 4 0.5 20 370 0.5 007 °26'04.6" 06°28'50.5" 5 2.5 25 374 1 007 °26'03.5" 06°28'50.4" 10 4 30 380 2.5 007 °26'05.3" 06°28'47.6" 5 2.5 28 360 1.6 007 °26'05.1" 06°28'47.6" 5 2 20 358 1 007 °26'05.2" 06°28'47.4" 2 0.15 24 355 0.3 007 °26'05.3" 06°28'47.3" 4 1.5 22 355 1 007 °26'06.9" 06°28'43.1" 10 5 22 348 2 007 °26'08.8" 06°28'41.7" 4 11 20 339 3 007 °26'10.1" 06°28'40.5" 4 1.5 20 330 0.3 007 °26'10.2" 06°28'40.6" 1.5 0.5 18 331 0.3 007 °26'11.7" 06°28'40.2" 1.3 0.6 15 322 0.2 Table 2 Summary of field measurements from the sedimentary terrain (Continued) Although liquefaction may be an associated mechanism of movement, the non-discrete nature of the shear zones and the short travel distances of majority of the slides make such conclusion uncertain. The Enugu landslides Figure 12 Correlation between frequency of landslide and slope angle indicating that landslides occur more on slopes with angles greater than 30 degree but less than 40 degree. season in October and early November. Landslides are fre- quent and widespread geomorphological phenomena the world over (Guzzetti, 1999; Dai et al. 2002; Das et al. 2010). Several people were killed and many more trapped when landslides were triggered by a heavy downpour in October 14th 2013 in the tourist town of Obudu that hosts thousands of foreign visitors annually. Helicopters rescued foreign and local people who were stranded inside this popular amusement and strategic arena. While early rains at the beginning of the wet season are usually the cause of most landslides in some parts of southeastern Nigeria, heavy rains during the peak season induced mass move- ments on the mountains of Obudu. There had been no sign of rain on the fateful day as tourist scanned the scen- ery. The sudden rain induced many landslides simultan- eously and prompted a rescue effort from the Municipal Government. warning systems aimed at preventing property damage and loss of human lives. The shallow sliding that result after a rainfall event in- volves the movement of debris, rock fragments, coarse to fine medium sands and the vegetation skin of the slope. Many landslides exhibit a combination of two or more types of movements, resulting in a complex type (Varnes 1984) and may be triggered by a number of exter- nal factors, such as intense rainfall, water level change, storm waves, rapid stream erosion etc. (Dai et al. 2002). Landslides are typical geomorphologic phenomena asso- ciated with the normal erosion cycle in tropical climates in hilly areas. They are frequent in some parts of Nigeria, and become disastrous when they affect populated areas or man-made structures. From the analyses, the materials from the Obudu, Calabar landslide are likely to be less porous than the materials from the Enugu landslide; and also have higher cohesion (Figure 13). Porosity, particle size, particle size distribution particle shape and orienta- tion, and stress history have significant effect on perme- ability (Selby 1993) which in turn exerts strong influence on the shear behavior of slope materials. These factors may have influenced the different mechanisms of failure in the two areas of interest. The Enugu landslides Sassa (1998) and Wang et al. (2002) noted that liquefaction may develop on similar materials but not at source but as motion progresses and modification and entrainment of mate- rials occur. One of the landslides with its lithologic se- quence is shown in Figure 10. The slide involved the sequence of coarse to medium friable sand units overly- ing fine to medium sand body inter-bedded with thin, impermeable claystone layers. Field observations indi- cated the existence of interlayer of claystone/shale in the debris directly below the displaced materials. The slope height and inclination were 67 m and 42° respect- ively. The slopes are characterized by well-defined un- dulating surfaces and landslide scarps. In rainy season, the slopes are covered by shrubs and grass. During the six months of dry season, partial stripping of the slopes occurs, decreasing the resistance of the slopes to ero- sion or landsliding. The return of rains subjects the area to intense gulling and surface failures. The water table varied from 7 to 15 m. Table 2 displays the results of field investigation on the sedimentary terrain where landslides are ubiquitous. Analysis of the 63 shows that most of the landslides have width < 7 m and length < 15 m (the slides are constrained to a particular narrow dimension), and that there is a particular trend of landslide occurrence in the area. The slides also occur more frequently on slopes which angles are between 30° and 35° (Figures 11 and 12). The trend appears to show that most of the landslides with bigger width also have larger length, and occur more on slopes with angles greater than 30° but less than 40°, which may be related to the triggering mechanism. It can be predicted therefore that the Enugu area is more prone to smaller slides than those with bigger dimensions. At every landslide width, the length could be determined, and vice versa. The prediction of potential landslide displace- ment and size should be a critical component of an early Figure 11 The relationship among landslide dimensions and slope angle. Figure 11 The relationship among landslide dimensions and slope angle. Page 11 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Figure 12 Correlation between frequency of landslide and slope angle indicating that landslides occur more on slopes with angles greater than 30 degree but less than 40 degree. The Enugu landslides In addition, the landslides at the metamorphic terrain have deeper depth to sliding sur- face than those of the sedimentary landscape. Using Cruden and Varnes (1996) the landslides in both lo- calities were characterized according to their types of move- ments, the materials involved and the states or activities of failed slopes. It was found that the two areas were domi- nated by debris slides. Montgomery and Dietrich (1994) have highlighted the importance of topographic control on shallow landslides. In the metamorphic terrain, the sliding material is dominated by finer fraction (71.5%) of micaceous silty sand and coarser fraction of gravel to boulder size. Sat- uration of the residual soil, weak bedrock and high slope angle are the primary factors in the slide-trigger. At the sedi- mentary area, the sliding mass is mainly loose, friable sands of high permeability. Saturation at the sand-shale interface Landsides in Iva Valley, Enugu are mainly triggered dur- ing the intense rains that mark the outset of wet season in April or May, while those in Obudu, Calabar occur mainly during the heavy rains that precede the end of rainy Page 12 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Figure 13 The shear strength parameters of Obudu and Iva Valley specimens. Figure 13 The shear strength parameters of Obudu and Iva Valley specimens. interest in the cost of landslide damage and thereby bring about heightened awareness on the need for im- proved strategies for safety. facilitates the formation of slip surface and eventual failure of the masses, which are quite consistent with Wu and Sidle (1995), Wilson and Wieczorek (1995) and Jakob et al. (2003). The research developed a potential prediction model for the size of landslide (Figure 11). The predic- tion of potential landslide displacement and size should be a critical component of an early warning systems aimed at preventing property damage and loss of human lives. References Aleotti P (2004) A warning system for rainfall-induced shallow failures. Eng Geol 73:247–265 Aleotti P, Chowdhury R (1999) Landslide hazard assessment: summary review and new perspectives. Bull Eng Geol Environ 58:21–44 new perspectives. Bull Eng Geol Environ 58:21–44 The sedimentary zone was more susceptible to landslid- ing than the areas that lay within the metamorphic locality. Evidently, there were more landslides on the sedimentary terrain despite a relatively low elevation range of 300 – 450 m. The climatic condition, deforestation, and erosion at the study area accelerate weathering and influence land- slide occurrence. During the peak of rains in October, land- slides are common in the international tourist area of Obudu and their mechanism depends upon the thickness of the loose residual soil. Chen Z, Wang J (2007) Landslide hazard mapping using logistic regression model in Mackenzie valley, Canada. Nat Hazards 42:75–89 Cruden D, Varnes DJ (1996) Landslide Types and Processes. In: Turner AK, Schuster RL (eds) Landslides Investigation and Mitigation. Special report 247 Transportation Research Board. National Academy of Sciences, Washington, DC, pp 36–75 Dai FC, Lee CF, Ngai YY (2002) Landslide risk assessment and management: an overview. Eng Geol 64(1):65–87 Das I, Sahoo S, Van Westen CJ, Stein A, Hack R (2010) Landslide susceptibility assessment using logistic regression and its comparison with a rock mass classification system, along a road section in the northern Himalayas (India). Geomorphology 114:627–637 Das I, Sahoo S, Van Westen CJ, Stein A, Hack R (2010) Landslide susceptibility assessment using logistic regression and its comparison with a rock mass classification system, along a road section in the northern Himalayas (India). Geomorphology 114:627–637 Edet AE, Teme SC, Okereke CS, Esu EO (1994) Lineament analysis for groundwater exploration in Precambrian Oban massif and obudu plateau, SE Nigeria. J Min Geol 30(1):87–95 Two failure mechanisms are proposed for the two ter- rains. For the metamorphic terrain with lower perme- ability but higher cohesion, stagnancy of rain on the material akin to prolonged rain infiltration produced a rise in pore pressure that initiated failure. The initial dis- placement following the failure probably weakened or destroyed the cohesion along a relatively deeper failure plane. Further rainfall during the same period triggered the sudden and rapid movement of debris downslope with movement only stopping because debris had crossed the road and hit the base of the opposite slope. Conclusions Field observations showed that the landslides on the steep metamorphic terrain have discrete, slickensided shear surfaces. There were rupture surfaces from which movements began as slides and continued for long dis- tances as mudflow/slides. The materials involved were typically poorly-sorted sandy colluviums supported by about 43% clay matrix produced from the weathering of the rocks. The characteristics the clay matrix probably con- tributed to the rapid movement and longer travel distance witnessed on the metamorphic terrain. On the sedimentary terrain, slumps and short runout debris slides of limited volume dominate the medium steep landscape. The slow to rapid mostly unsaturated movements usually initiate within the sandy body or on clayey/shale interlayer, but are It is noted that extensive human interference in hill slope areas for construction of roads along the hill slopes, deforestation, and change in land use have contribute to instability in the Obudu area. Communication with policy makers and planners can avert disaster. Correct develop- ment planning is necessary to avoid irreparable mistakes especially where risks are high and safety of lives and property are the key issues. The social and economic costs of landslides are not well documented because of the dearth of scientific and inventory data. It is hoped that our study will stimulate Page 13 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Received: 3 May 2014 Accepted: 21 October 2014 References For the slopes on the sedimentary terrain with little cohe- sion, the high permeability of the materials ensured signifi- cant pore pressure was developed at the sand-shale interface. Additional stress from rain and self-weight read- ily initiates much shallower failures that may move a few meters because of the absence of satisfactory conditions that could result in a transformation to flow. The occur- rence of landslides in Iva Valley Enugu can be correlated to slope gradient and vegetation cover. Most failures on the sandy slopes, which were < 1 m deep, occurred on slopes with gradients between 30 and 35. Ejimofor OC, Umeji AC, Turaki UM (1996) Petrology and major element geochemistry of the basement rocks of northern Obudu area, eastern Nigeria. J Min Geol 32:1–9 Igwe O, Mode W, Nnebedum O, Okonkwo I, Oha I (2013) The analysis of rainfall- induced slope failures at Iva Valley area of Enugu State, Nigeria. Environ Earth Sci doi:10.1007/s12665-013-2647-x Iverson RM (2000) Landslide triggering by rain infiltration. Water Resour Res 36(7):1897–1910 Iverson RM (2000) Landslide triggering by rain infiltration. Water Resour Res 36(7):1897–1910 Jakob M, Weatherly H (2003) A hydroclimatic threshold for landslide initiation on the North Shore Mountains of Vancouver, British Columbia. Geomorphology 54:137–156 Jakob M, Weatherly H (2003) A hydroclimatic threshold for landslide initiation on the North Shore Mountains of Vancouver, British Columbia. Geomorphology 54:137–156 Kogbe CA (1989) Palaeogeographic History of Nigeria from Albian times. In: Kogbe CA (ed) Geology of Nigeria. Elizabethan Publ.Co, Lagos, pp 257–275 Kogbe CA (ed) Geology of Nigeria. Elizabethan Publ.Co, Lagos, pp 257–275 Liao ZH, Hong Y, Kirschbaum D, Liu C (2012) Assessment of shallow landslides from Hurricane Mitch in central America using a physically based model. Environ Earth Sci 66(6):1697–1705 Marchi L, Arattano M, Deganutti AM (2002) Ten years of debrisflow monitoring in the moscardo torrent (Italian alps). Geomorphology 46:1–17 Montgomery DR, Dietrich WE (1994) A physically based model for the topographic control of shallow landsliding. Water Resour Res 30(4):1153–1171 Msilimba G, Holmes P (2010) Landslides in the rumphi district of northern Malawi: characteristics and mechanisms of generation. Nat Hazards 54(3):657–677 Received: 3 May 2014 Accepted: 21 October 2014 typically on non-discrete, matrix-poor shear surfaces. The movements are mainly non-confined and without estab- lished channel. Generally, the size of the shallow landslides appeared to depend on soil thickness and saturation. The landslides occurred when soil thickness accumulating at sufficient depth was saturated by rain infiltration. Consent Liao ZH, Hong Y, Kirschbaum D, Liu C (2012) Assessment of shallow landslides from Hurricane Mitch in central America using a physically based model. Environ Earth Sci 66(6):1697–1705 Written informed consent was obtained from the (persons whose images are present in Figures 1 and 8) for the publication of this report and any accompanying images. Montgomery DR, Dietrich WE (1994) A physically based model for the topographic control of shallow landsliding. Water Resour Res 30(4):1153–1171 Msilimba G, Holmes P (2010) Landslides in the rumphi district of northern Malawi: characteristics and mechanisms of generation. Nat Hazards 54(3):657–677 Competing interests The author declares that he has no competing interests. Competing interests The author declares that he has no competing interests. Acknowledgement h h Oboh-Ikuenobe FE, Obi GC, Jamarillo CA (2005) Lithofacies, palynofacies and sequence stratigraphy of paleogene strata in Southeastern Nigeria. J Afr Earth Sci 41:79–101 The research was carried under the auspices of the Engineering and Environmental Geology Group of the University of Nigeria, Nsukka and was supported by the President of the University of Nigeria who encouraged research as the major development platform in the institution. I am indebted to all staff and students of the Department of Geology, University of Nigeria, Nsukka for the useful aid they offered during the field, laboratory and analytical stages of this work. Petrucci O, Polemio M (2009) The role of meteorological and climatic conditions in the occurrence of damaging hydro-geologic events in Southern Italy. Nat Hazards Earth Syst Sci 9:105–118 Sassa K (1998) Mechanisms of landslide triggered debris flows. Environ Forest Sci 54:499–518 Sassa K (1998) Mechanisms of landslide triggered debris flows. Environ Forest Sci 54:499–518 Page 14 of 14 Igwe Geoenvironmental Disasters (2015) 2:1 Sassa K, Wang G, Fukuoka H, Wang FW, Ochiai T, Sugiyama ST (2004) Landslide risk evaluation and hazard mapping for rapid and long-travel landslides in urban development areas. Landslides 1(3):221–235 Selby MJ (1993) Hillslope Materials and Processes, 2nd edn. Oxford University Press, New York, p 451 Toteu SF, Penaye J, Djomani YP (2004) Geodynamic evolution of the Pan-African belt in central Africa with special reference to Cameroon. Can J Earth Sci 41:73–75 Ukaegbu VU, Beka FT (2007) Petrochemistry and geotectonic significance of enderbite-charnockite association in the Pan-African Obudu plateau, southeastern Nigeria. J Min Geol 43(1):1–14 southeastern Nigeria. J Min Geol 43(1):1–14 Ukaegbu VU, Oti MN (2004) Geology of the Pan-African metamorphic rocks of southern obudu plateau, bamenda massif, southeastern Nigeria. Scientia Africana 3(2):8–18 Varnes DJ (1984) Landslide Hazard Zonation: a Review of Principles and Practice Review report. UNESCO, Daramtiere, Paris, p 61 Varnes DJ (1984) Landslide Hazard Zonation: a Review Review report. UNESCO, Daramtiere, Paris, p 61 Varnes DJ (1984) Landslide Hazard Zonation: a Revie Review report. UNESCO, Daramtiere, Paris, p 61 Wang FW, Sassa K (2010) Landslide simulation by a geotechnical model combined with a model for apparent friction change. Phys Chem Earth 35(3–5):149–161 Wang FW, Sassa K (2010) Landslide simulation by a geotechnical model combined with a model for apparent friction change. Phys Chem Earth 35(3–5):149–161 Wang FW, Sassa K, Fukuoka H (2000) Geotechnical simulation test for the Nikawa landslide induced by January 17, 1995 Hyogoken-Nambu earthquake. Acknowledgement h h Soils Found 40(1):35–46 Wang FW, Sassa K, Wang G (2002) Mechanism of a long-runout landslide triggered by the August 1998 heavy rainfall in Fukushima prefecture, Japan. Eng Geol 63(1–2):169–185 Wang FW, Matsumoto T, Tanaka Y (2005) Two recent flowslides in Yamashina area, Kanazawa city, Japan. Landslides 2(3):229–234 Wang FW, Cheng Q, Highland L, Wang H, Yan C (2009) Preliminary investigation of some large landslides triggered by the 2008 Wenchuan earthquake, Sichuan Province, China. Landslides 6(1):47–54 Wei NP, Nutalaya P, Aung Z, Zhibin T (1993) Catastrophic landslides and debris flows in Thailand. Bull Int Assoc Eng Geol 48(1):93–100 Wieczorek GF, Glade T (2005) Climatic Factors Influencing Occurrence of Debris Flows. In: Jakob M, Hungr O (eds) Debris Flow Hazards and Related Phenomena. Springer, Berlin Heidelberg, pp 325–362 Wilson RC, Wieczorek GF (1995) Rainfall thresholds for the initiation of debris flow at La Honda, California, Environ. Eng Geosci 1(1):11–27 Wu W, Sidle RC (1995) A distributed slope stability model for steep forested basins. Water Resour Res 31:2097–2110 Wang FW, Sassa K, Fukuoka H (2000) Geotechnical simulation test for the Nikawa landslide induced by January 17, 1995 Hyogoken-Nambu earthquake. Soils Found 40(1):35–46 Wang FW, Sassa K, Wang G (2002) Mechanism of a long-runout landslide triggered by the August 1998 heavy rainfall in Fukushima prefecture, Japan. Eng Geol 63(1–2):169–185 Wang FW, Matsumoto T, Tanaka Y (2005) Two recent flowslides in Yamashina area, Kanazawa city, Japan. Landslides 2(3):229–234 Submit your manuscript to a journal and beneﬁ t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the ﬁ eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com
https://openalex.org/W2125620273	https://uobrep.openrepository.com/bitstream/10547/296166/2/1741-7015-11-29.pdf	English	null	Paternal obesity is associated with IGF2hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort	BMC medicine	2,013	cc-by	9,132	© 2013 Soubry et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: adelheid.soubry@hotmail.com 1Duke Cancer Institute, Duke University Medical Center 2715, Durham, NC 27710, USA Full list of author information is available at the end of the article Soubry et al. BMC Medicine 2013, 11:29 Soubry et al. BMC Medicine 2013, 11:29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort Adelheid Soubry1*, Joellen M Schildkraut1,2, Amy Murtha3, Frances Wang1, Zhiqing Huang4, Autumn Bernal5, Joanne Kurtzberg6, Randy L Jirtle5, Susan K Murphy4 and Cathrine Hoyo7 See related commentary article here http://www.biomedcentral.com/1741-7015/11/30 e related commentary article here http://www.biomedcentral.com/1741-7015/11/30 Background Environmental exposures acquired early in life have been correlated with persistent modifications of the epigen- ome. The epigenetic information in human cells is stored via mitotically heritable DNA methylation, organization of the chromatin structure (for example, histone modifi- cation), and regulatory RNAs. Together, these mechan- isms are responsible for regulating gene expression during cellular differentiation during embryonic develop- ment and throughout life [1]. Our study focuses on the DNA methylation patterns of the imprinted Insulin-Like Growth Factor 2 (IGF2) gene, coding a well-characterized growth factor active throughout embryogenesis and fetal growth [2,3]. In normal human tissues only the paternal IGF2 allele is transcribed; and its imprinting is regulated by at least two differentially methylated regions (DMRs): one is located upstream of the three IGF2 promoters that are subject to imprinting (IGF2 DMR), and the other is located upstream of the neighboring non-coding H19 gene (H19 DMR). The latter region is part of the imprint- ing control region (ICR) which harbors binding sites for the zinc finger protein CTCF. During early development, imprint marks are erased in the primordial germ cells and new methylation imprints are established according to the germ cell. This has particularly been demonstrated at the IGF2 DMR [4] and H19 DMR [4,5] in sperma- togenic human cell stages. The progressive imprint re-establishment of the DNA methylation imprint marks throughout human spermatogenesis leads to fully methy- lated IGF2 and H19 DMRs. Consequently, methylation is only present on the paternally inherited allele in the off- spring. Shifts in methylation established at these DMRs can lead to loss of imprinting and transcription of IGF2 may be altered [6,7]. Hence, normal physiological mechanisms or homeostasis in the body may be skewed and lead to chronic diseases later in life. Until now, epi- demiological studies have focused on maternal factors and especially in utero exposures to certain nutritional or environmental conditions as the potential explanation for such disruptions or shifts in methylation at the DMRs [8-10]. This can potentially contribute to a higher risk for obesity [11], chronic diseases at later age [12], including diabetes or cardiovascular diseases [13-15], or even can- cer [16,17]. Animal experiments show that modification of maternal diet during development can influence meta- bolism in adulthood [18]. Abstract BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Page 2 of 10 Although paternal obesity is equally prevalent, the major- ity of the epidemiological studies to date suggest in utero exposures as the only possible origin of potential epige- nomic modifications at birth. Obesity is associated with over-nutrition, unbalanced food intake (such as low vege- table consumption), and a sedentary lifestyle [22]. Conse- quently, elucidating the epigenetic risks associated with the current “Western” lifestyle on the next generations is crucial. In the current report we determined whether preconceptional obesity, in the mother or the father, is associated with methylation patterns at the IGF2 DMRs in newborns using DNA from leukocytes isolated from umbilical cord blood at birth. By including paternal obe- sity in our study we were able to examine a potential pre- conceptional impact of the environment on imprint mark reprogramming during male gametogenesis. Conse- quently, we found that paternal obesity is associated with a decrease in DNA methylation at the IGF2 DMR. Data collection Th i The questionnaire included maternal socio-demographic data, such as age, marital status, race and education, as well as multiple lifestyle or health characteristics (for example, smoking, chronic diseases). One of the main items of the survey included a detailed list of questions about the mother’s and the father’s height, highest and lowest weight ever, current and usual weight. Medical records were used to verify medical conditions, and to abstract gestation time, birth weight and the newborn’s gender. Body mass index was calculated from the data obtained from height and the mother’s weight before pregnancy or the father’s current weight. Obesity was defined as BMI ≥30 kg/m2. Background Although the underlying mechanism and crucial time points of exposure are not clear, changes in epigenetic regulation are now regarded as a highly plausible explanation for linking the associa- tions between dietary exposures in early life to the onset of chronic diseases during adulthood. Several lines of evi- dence suggest that pre- or periconceptional obesity of the mother may affect metabolic programming [19-21]. Study participants We studied a subgroup of the first 98 families enrolled in the Newborn Epigenetics Study cohort. Seventy-eight mothers provided detailed information about the biolo- gical fathers. One mother gave birth to twins, bringing the total number of newborns with corresponding pater- nal data to 79. The enrollment process was assisted by a trained interviewer. Detailed recruitment strategies for NEST have been described in a previous study [23]. English speaking pregnant mothers delivered between July 2005 and November 2006 at Duke University Hos- pital, Durham, NC. Abstract Background: Data from epidemiological and animal model studies suggest that nutrition during pregnancy may affect the health status of subsequent generations. These transgenerational effects are now being explained by disruptions at the level of the epigenetic machinery. Besides in vitro environmental exposures, the possible impact on the reprogramming of methylation profiles at imprinted genes at a much earlier time point, such as during spermatogenesis or oogenesis, has not previously been considered. In this study, our aim was to determine associations between preconceptional obesity and DNA methylation profiles in the offspring, particularly at the differentially methylated regions (DMRs) of the imprinted Insulin-like Growth Factor 2 (IGF2) gene. Methods: We examined DNA from umbilical cord blood leukocytes from 79 newborns, born between July 2005 and November 2006 at Duke University Hospital, Durham, NC. Their mothers participated in the Newborn Epigenetics Study (NEST) during pregnancy. Parental characteristics were obtained via standardized questionnaires and medical records. DNA methylation patterns at two DMRs were analyzed by bisulfite pyrosequencing; one DMR upstream of IGF2 (IGF2 DMR), and one DMR upstream of the neighboring H19 gene (H19 DMR). Multiple regression models were used to determine potential associations between the offspring’s DNA methylation patterns and parental obesity before conception. Obesity was defined as body mass index (BMI) ≥30 kg/m2. Results: Hypomethylation at the IGF2 DMR was associated with paternal obesity. Even after adjusting for several maternal and newborn characteristics, we observed a persistent inverse association between DNA methylation in the offspring and paternal obesity (b-coefficient was -5.28, P = 0.003). At the H19 DMR, no significant associations were detected between methylation patterns and paternal obesity. Our data suggest an increase in DNA methylation at the IGF2 and H19 DMRs among newborns from obese mothers, but a larger study is warranted to further explore the potential effects of maternal obesity or lifestyle on the offspring’s epigenome. Conclusions: While our small sample size is limited, our data indicate a preconceptional impact of paternal obesity on the reprogramming of imprint marks during spermatogenesis. Given the biological importance of imprinting fidelity, our study provides evidence for transgenerational effects of paternal obesity that may influence the offspring’s future health status. : Epigenetics, DNA methylation, IGF2, obesity, offspring, Newborn Epigenetics Study, Epidemiology Keywords: Epigenetics, DNA methylation, IGF2, obesity, offspring, Newborn Epigenetics Stud Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. Specimen collection At delivery, umbilical cord blood samples were collected in a vacuum blood collection tube coated with K3EDTA. The tubes were centrifuged to isolate the buffy coat; this leukocyte-containing buffy coat was then stored at Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Page 3 of 10 Table 1 Parental and newborn characteristics NEST - Newborn Epigenetics Study Cohort 2005 to 2006 n % BMI mother: BMI <30 (not obese) 59 67.8 BMI ≥30 (obese) 28 32.2 BMI father: BMI <30 (not obese) 63 79.7 BMI ≥30 (obese) 16 20.3 Marital status: Living with partner 72 74.2 Single 25 25.8 Education: Low (no college degree) 57 58.8 High (at least college degree) 40 41.2 Race: African American 38 38.8 Caucasian 56 57.1 Other or not specified 4 4.08 Maternal age: <30 years 56 57.1 ≥30 years 42 42.9 Smoking: Mother never smoked 45 48.4 Quit smoking when pregnant 26 27.9 Smoked during pregnancy 22 23.7 Gestation time: Preterm (<37 weeks) 10 10.3 Normal (≥37 weeks) 87 86.7 Birth weight: <2.5 kg 16 16.5 ≥2.5 kg 81 83.5 Baby gender: Male 48 49.5 Female 49 50.5 This sub-cohort includes all NEST families from whom babies were born at Duke University Hospital between July 2005 and November 2006. Characteristics of mothers, fathers and newborns are shown for the 98 participants when data were not missing. -80ºC. Genomic DNA was extracted using Gentra Pure- gene Reagents (Qiagen, Valencia, CA, USA). -80ºC. Genomic DNA was extracted using Gentra Pure- gene Reagents (Qiagen, Valencia, CA, USA). Table 1 Parental and newborn characteristics NEST - Newborn Epigenetics Study Cohort 2005 to 2006 Table 1 Parental and newborn characteristics DNA methylation analysis y y IGF2 and H19 DMRs were analyzed by pyrosequencing. The IGF2 DMR includes three CpG dinucleotides upstream of IGF2 exon 3 (chr 11p15.5; CpG site 1: 2,169,518; CpG site 2: 2,169,515; and CpG site 3: 2,169,499; NCBI Human Genome Build 37/hg19). This DMR has been previously evaluated by Cui et al. [6] and Heijmans et al. [8]. The region studied for the H19 DMR encompasses four dinucleotides located upstream of the H19 gene (chr 11p15.5; CpG site 1: 2,024,261, CpG site 2: 2,024,259, CpG site 3: 2,024,257, and CpG site 4: 2,024,254; NCBI Human Genome Build 37/hg19), which is the first of six known sequence motifs that bind the CTCF zinc finger protein [24,25]. The struc- tural characteristics of these IGF2 and H19 loci and the genomic coordinates of the assays have been presented elsewhere [7,26]. Genomic DNA (800 ng) was treated with sodium bisulfite [27], the IGF2/H19 regions were amplified by PCR, and pyrosequencing was performed in duplicate using a Pyromark Q96 MD pyrosequencing instrument (Qiagen). Control assays were also run to validate our methylation results. Detailed methodology, including assay conditions and validation studies, have been described previously [25]. This sub-cohort includes all NEST families from whom babies were born at Duke University Hospital between July 2005 and November 2006. Characteristics of mothers, fathers and newborns are shown for the 98 participants when data were not missing. This sub-cohort includes all NEST families from whom babies were born at Duke University Hospital between July 2005 and November 2006. Characteristics of mothers, fathers and newborns are shown for the 98 participants when data were not missing. Statistical methods We evalu- ated potential confounding for paternal and maternal obe- sity by means of multivariable regression analyses. We used multiple regression analyses, separately for each exposure and characteristics from Table 1. These multiple regression analyses were used to predict DNA methylation at each DMR site. To further explore whether the associa- tion between obesity and DNA methylation varied by race, we repeated these analyses in African Americans and Cau- casians. Graphs representing the associations between BMI and methylation outcomes were based on the pre- dicted outcomes of the methylation means, after adjusting for maternal age, smoking status, birth weight and gender. Observations included in the regression analyses were determined by the availability of laboratory measures for the IGF2 and H19 DMRs. All statistical analyses were con- ducted in SAS v9.2 (SAS Institute Inc., Cary, NC, USA), and GraphPad Prism 5 was used to obtain Figures 1 and 2 (GraphPad Software Inc., La Jolla, CA, USA). Statistical methods Variables were defined as follows: race (African American, Caucasian, and other), age (as a continuous variable), marital status (living with partner versus single), at least a college graduate (yes or no), pre-pregnancy maternal and paternal obesity (<30 versus ≥30 kg/m2), smoking status (never/quit smoking when pregnant/continued smoking during pregnancy), birth weight of the baby (<2.5 kg/ between 2.5 kg and 3.5 kg/≥3.5 kg), gestation time (<37 weeks versus ≥37 weeks), and gender of the baby (male or female) (Table 1). Chi Square tests were used to compare obesity of the mother and the father within different sub- groups of pregnant women. If numbers were small (<5) the Fisher exact test was used. We also compared the characteristics for missing and non-missing experimental methylation results and for missing and non-missing BMI of the father. Methylation levels were distributed normally in the groups studied (verified by using the Kolmogorov- Smirnov test). Student’s t-tests were computed to test for significant differences in the methylation means. We assessed the effect of maternal and paternal obesity (BMI ≥30 kg/m2) on the methylation levels of the IGF2 and H19 DMRs, which were analyzed separately by individual CpG site, as well as by the mean of the CpGs. To account for batch effects from the laboratory tests on different days or different plates we calculated the least square means (or estimated marginal means) of each CpG site. We evalu- ated potential confounding for paternal and maternal obe- sity by means of multivariable regression analyses. We used multiple regression analyses, separately for each exposure and characteristics from Table 1. These multiple regression analyses were used to predict DNA methylation at each DMR site. To further explore whether the associa- tion between obesity and DNA methylation varied by race, we repeated these analyses in African Americans and Cau- casians. Graphs representing the associations between BMI and methylation outcomes were based on the pre- dicted outcomes of the methylation means, after adjusting for maternal age, smoking status, birth weight and gender. Observations included in the regression analyses were determined by the availability of laboratory measures for the IGF2 and H19 DMRs. All statistical analyses were con- ducted in SAS v9.2 (SAS Institute Inc., Cary, NC, USA), and GraphPad Prism 5 was used to obtain Figures 1 and 2 (GraphPad Software Inc., La Jolla, CA, USA). estimated marginal means) of each CpG site. Ethics The experimental research in this report was performed with the approval of the Duke University Institutional Review Board, reference number: Pro00014548; this means that the research carried out is in compliance with Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015 Page 4 of 10 Soubry et al. BMC Medicine 2013, 11:29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 http://www.biomedcentral.com/1741-7015/11/29 Figure 1 Methylation at the IGF2 and H19 DMRs in the offspring by parental obesity. The graphs represent the mean estimated methylation values of 69 newborns at the IGF2 DMR, and 70 newborns at the H19 DMR. The IGF2 DMR results are based on 14 obese fathers and 25 obese mothers; the results at the H19 DMR are based on 15 obese fathers and 23 obese mothers. For each exposure the differences of the least square means of methylation percentages are shown at each CpG site (bold), as well as standard errors (SE) and P-values. Bars represent standard errors. Figure 1 Methylation at the IGF2 and H19 DMRs in the offspring by parental obesity. The graphs represent the mean estimated methylation values of 69 newborns at the IGF2 DMR, and 70 newborns at the H19 DMR. The IGF2 DMR results are based on 14 obese fathers and 25 obese mothers; the results at the H19 DMR are based on 15 obese fathers and 23 obese mothers. For each exposure the differences of the least square means of methylation percentages are shown at each CpG site (bold), as well as standard errors (SE) and P-values. Bars represent standard errors. during pregnancy, and 32% were obese before pregnancy. About 80% of the mothers provided data about the father’s characteristics: 20% are obese. Approximately 16% of the newborns had a low birth weight, 10% was born preterm and gender was equally distributed. None of the included characteristics were associated with paternal obesity, with the exception of obesity in the mother (P = 0.01). For instance, potential associations between birth weight and parental obesity were excluded by Chi Square tests or Fisher exact tests; P-values were 0.7 for paternal and maternal obesity. Maternal obesity was positively associated with single motherhood (P = 0.002), being African American (P <0.001) and being a non-smoker (P = 0.06). Ethics Given that 19% of our subjects did not report detailed information about the fathers, we compared all measured characteristics and methylation end-results in newborns with missing anthropometric the Helsinki Declaration. All participants signed the informed consent for the use of their medical record data, questionnaire data, their newborn’s birth para- meters and umbilical cord blood. Characteristics of study participants The distributions of socio-demographic data are shown in Table 1. Seventy-four percent of the mothers were liv- ing with a partner, and nearly 26% were single. The majority of the study population were Caucasians (57.1%) or African Americans (38.8%), and the other races/ethni- cities included Asians, Native Americans or non-specified race (4.1%). A quarter of the mothers were between 25 and 29 years old (26.5%). Nearly 59% never obtained a college degree, and were categorized as “low education”. Approximately 24% of the mothers reported smoking Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. BMC Medicine 2013, 11:29 Page 5 of 10 http://www.biomedcentral.com/1741-7015/11/29 Figure 2 Offspring’s mean methylation % at the IGF2 DMR by BMI of the parents. The predicted methylation means at IGF2 DMR are plotted by BMI of the father (upper graph), and BMI of the mother (lower graph); adjusted for maternal age, smoking status, BMI of the other parent, the newborn’s birth weight and gender. Figure 2 Offspring’s mean methylation % at the IGF2 DMR by BMI of the parents. The predicted methylation means at IGF2 DMR are plotted by BMI of the father (upper graph), and BMI of the mother (lower graph); adjusted for maternal age, smoking status, BMI of the other parent the newborn’s birth weight and gender Figure 2 Offspring’s mean methylation % at the IGF2 DMR by BMI of the parents. The predicted methylation means at IGF2 DMR are plotted by BMI of the father (upper graph), and BMI of the mother (lower graph); adjusted for maternal age, smoking status, BMI of the other parent, the newborn’s birth weight and gender. DMRs in our cohort. Figure 1 shows the estimated means of methylation percentages by obesity of the parents. Differences in methylation percentages among offspring from obese fathers compared to non-obese fathers at CpG1, CpG2, and CpG3 of the IGF2 DMR were: -3.36% (P = 0.08), -3.56% (P = 0.01) and -3.57% (P = 0.02), respec- tively; the mean methylation difference was: -3.50% (P = 0.02) (Figure 1). Methylation in offspring among obese fathers was significantly lower than when compared to fathers who were not obese. We observed no differences in methylation percentages at the IGF2 DMR between off- spring of obese and non-obese mothers. Characteristics of study participants Differences in methylation percentages at CpG1, CpG2 and CpG3 were +2.09% (P = 0.18), +0.66% (P = 0.57) and +0.90% (P = 0.44), respectively; the mean methylation difference was +1.19% (P = 0.32) (Figure 1). data about fathers versus those without missing data. The majority of the missing paternal data (78%) were from African American mothers with lower education. Single mothers were less likely to answer questions about the father’s weight and height (32.0%), compared with mar- ried mothers or those living with a partner (13.9%), P = 0.07. Other characteristics and the methylation outcomes did not differ significantly between both groups. Associations between paternal or maternal obesity and methylation profiles at the IGF2 and H19 DMRs in the offspring Associations between paternal or maternal obesity and methylation profiles at the IGF2 and H19 DMRs in the offspring p g Using DNA extracted from umbilical cord blood leuko- cytes we determined the levels of methylation at CpG sites within the IGF2 and H19 DMRs in newborns. From the 79 samples in our cohort, we obtained experimental data for 67 participants at the IGF2 DMR and for 70 partici- pants at the H19 DMR. We further refer to these partici- pants as our analytical cohort. No significant differences in characteristics were found when comparing the missing with the non-missing experimental methylation results for both DMRs. We analyzed methylation outcomes by the characteristics described in Table 1 and with the exception of obesity (BMI ≥30 kg/m2), none showed significant asso- ciations with methylation percentages at the IGF2 or H19 At the H19 DMR we found no methylation differences in the offspring of obese compared to non-obese fathers. Differences in methylation percentages at CpG1, CpG2, CpG3 and CpG4 of the H19 DMR were +1.95% (P = 0.37), +1.68% (P = 0.42), +1.75% (P = 0.38) and +1.49% (P = 0.48), respectively; the mean methylation difference was +1.74% (P = 0.40) (Figure 1). However, the methyla- tion percentages at the CpG sites of the H19 DMR among offspring from obese mothers were significantly Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Page 6 of 10 (P = 0.07), and +2.80 when the mothers were obese (P = 0.05). The multivariate model with the mean methylation percent as a dependent variable and several measured maternal, paternal and newborn characteris- tics as independent variables (Model 3) showed a b- coefficient for offspring from obese fathers of +2.55 (P = 0.17), and a b-coefficient for offspring from obese mothers of +1.05 (P = 0.54). Similar results were seen when we evaluated the individual CpG sites (data not shown). higher at CpG1, CpG2, CpG3 and CpG4 compared to offspring from non-obese mothers. The differences were +3.56% (P = 0.04), +4.13% (P = 0.01), +4.08% (P = 0.01) and +4.55% (P = 0.007), respectively; the mean methyla- tion difference was +4.10% (P = 0.01) (Figure 1). In brief, without adjusting for potential confounders we detected significantly lower methylation at the IGF2 DMR asso- ciated with paternal obesity, and significantly higher methylation at the H19 DMR when the mother was obese. Associations between paternal or maternal obesity and methylation profiles at the IGF2 and H19 DMRs in the offspring To assess the independent effects of preconceptional maternal or paternal obesity on DNA methylation at the IGF2 and H19 DMRs in newborns, we used multiple regression models, controlling for potential confounders, including maternal age, maternal smoking status, gender of the baby, birth weight and experimental batch effects. The results of the regression analyses, found in Table 2, show that paternal obesity is inversely associated with DNA methylation levels at the IGF2 DMR in newborns. Controlling for potential confounding did not change this relationship. After adjusting for several characteris- tics, we observed a significant decrease in methylation when fathers were obese, b-coefficient was -5.28 (P = 0.003) (Table 2, Model 3). Little difference for this asso- ciation was observed when comparing the individual CpG sites. The b-coefficients for CpG1, CpG2 and CpG3 were -5.78 (P = 0.01), -5.18 (P = 0.002) and -4.76 (P = 0.007), respectively. In contrast, results for maternal obesity indicated an increase in methylation at the IGF2 DMR, the b-coefficient was +3.08 (P = 0.05) when con- trolling for the same characteristics (Table 2, Model 3). The b-coefficients at the individual CpG-sites were +4.23 (P = 0.04), +2.44 (P = 0.08), +2.42 (P = 0.12), for CpG1, CpG2 and CpG3; with only CpG1 reaching sta- tistical significance. We further stratified our analytical cohort by race (Caucasian and African American) and repeated all regression analyses described above. At the IGF2 DMR, the outcome was similar as in our complete analytical cohort. We calculated a b-coefficient of -5.16 in offspring from obese fathers, among Caucasians (P = 0.01) (as in Model 3); and a b-coefficient of -3.65 in offspring from obese fathers, among African Americans. Although the estimates suggest an inverse relationship in Caucasian subjects, this effect was not significant among African Americans (P = 0.34). These observations were based on only 10 obese fathers in Caucasians, and only 4 obese fathers in African Americans. At the H19 DMR, results were also similar and in the same direction as those in our complete analytical cohort. However, we found one higher estimate in Model 1 for children from African American obese fathers: b-coefficient was +8.14 (P = 0.007). But, this result was based on a small sample size with only 6 obese African American fathers. Together with our statistical power calculations we conclude that the stratified analyses represent unstable estimates. Associations between paternal or maternal obesity and methylation profiles at the IGF2 and H19 DMRs in the offspring We further extended the regression analyses in our analytical cohort by replacing the dichotomous obesity variable by the continuous BMI variable. At the H19 DMR, we found a b-coefficient of +0.21 (P = 0.12) for paternal BMI and +0.16 (P = 0.09) for maternal BMI, controlling for the same variables as before; but these results were still not significant. Whereas at the IGF2 DMR, our data reached a significant positive association when both paternal and maternal BMI were used as inde- pendent variables in the multivariate model; b-coeffi- cients were -0.38 (P = 0.009), and +0.22 (P = 0.02), respectively. These correlations between parental BMI and the predicted mean methylation levels at the IGF2 DMR in the offspring are depicted in Figure 2. In our separate regression models by maternal and paternal obesity, we observed a positive trend in DNA methylation at the H19 DMR when adjusting for mater- nal and newborn characteristics (Table 2, Models 1 and 2, respectively). The b-coefficient was +3.09 when the fathers were obese, but this result was not significant Table 2 Linear Regression Models: methylation at the IGF2 and H19 DMRs in relation to parental obesity Table 2 Linear Regression Models: methylation at the IGF2 and H19 DMRs in relation to parental obesity Linear regression models IGF2 DMR H19 DMR Obesity of: b SE P b SE P Model 1 Father -3.83 1.48 0.01 +3.09 1.64 0.07 Model 2 Mother +2.38 1.30 0.08 +2.80 1.38 0.05 Model 3 Father Mother -5.28 +3.08 1.62 1.48 0.003 0.05 +2.55 +1.05 1.82 1.70 0.17 0.54 Obesity was defined as BMI ≥30 kg/m2. All models were adjusted for maternal age and smoking status, as well as by the newborn’s birth weight and gender. Models 1 and 2 include either maternal or paternal obesity. Model 3 includes both maternal and paternal obesity. Discussion We explored the potential effect of parental obesity on IGF2/H19 DMR methylation in newborns. We found a significant decrease in methylation among newborns of obese fathers at the IGF2 DMR in DNA extracted from cord blood leucocytes. This finding remained significant after controlling for potential confounders (b-coefficient = -5.28, P = 0.003). Hypomethylation at the IGF2 DMR has Obesity was defined as BMI ≥30 kg/m2. All models were adjusted for maternal age and smoking status, as well as by the newborn’s birth weight and gender. Models 1 and 2 include either maternal or paternal obesity. Model 3 includes both maternal and paternal obesity. Page 7 of 10 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 been associated with an increased risk of developing can- cers, such as Wilms’ tumor [28], colorectal cancer [6] and ovarian cancer [7]. We found no significant changes in methylation levels associated with paternal obesity at the H19 DMR region. been associated with an increased risk of developing can- cers, such as Wilms’ tumor [28], colorectal cancer [6] and ovarian cancer [7]. We found no significant changes in methylation levels associated with paternal obesity at the H19 DMR region. insulin secretion, altered expression of multiple genes related to normal pancreatic beta-cell function, and altered methylation at a putative regulatory region of the Interleukin 13 receptor, alpha 2 gene [42]. Male mice whose mothers were exposed to a high-fat diet were not only obese, insulin insensitive and diabetic, they were also capable of passing part of this phenotype to the next generation, suggesting an underlying epigenetic mechan- ism transmitted through germ cells [43]. To our knowl- edge, our study is the first epidemiological study that suggests a similar underlying epigenetic mechanism con- ferred by harmful paternal dietary patterns or obesity. Obesity is a metabolic condition that has paradoxically been associated with poverty, low quality of life, malnu- trition and an imbalanced intake of nutrients [21,22]. Few epidemiological studies indicate potential correla- tions between obesity or food supplies in the paternal line and offspring’s birth weight [29], body-fat in prepu- bertal girls [30], or mortality from chronic diseases [31-33]. Epidemiological data regarding associations between maternal obesity and the offspring’s birth weight vary by study (reviewed by McDonald et al. [34]). We examined possible associations between paternal or maternal obesity and birth weight but detected no asso- ciations. Discussion Epidemiological studies on maternal obesity- related exposures generally show a positive association between abnormally high BMI and congenital anomalies (reviewed by Stothard et al. [35]). These harmful effects are mostly attributed to in utero exposures to malnutri- tion or overnutrition; while very often, data indicate the importance of exposures at the very early stages of devel- opment, even before conception. Research in animal models suggests that potential diet-dependent transge- nerational effects may be explained by changes in the establishment of epigenetic gene regulatory marks [12,36-38]. Analyses of adults born to mothers exposed to poor nutrition during the Dutch famine indicated a 5% decrease in methylation at the IGF2 DMR compared to the same sex non-exposed siblings. Interestingly, the magnitude of this effect is similar to the effect we observe in offspring from obese fathers at the same locus. In the Dutch famine cohort, exposure during the periconcep- tional period was more important than during the second or the third trimester of gestation [8]. Furthermore, not only do exposures during early gestation cause harmful health outcomes, but famine prior to conception has also been associated with poor health [39]. Similar results were found among Gambian children; they exhibit altered DNA methylation at several metastable epialleles according to the seasonal nutritional circumstances in which they were conceived [40]. Both cohorts described above did not directly examine the relationship with the fathers’ dietary patterns; although they were most likely exposed to the same famine or nutritional circumstances as the mothers. Analysis of the Framingham Heart Study indicates that early-onset paternal obesity, and not maternal obesity, increases the odds of aberrant serum levels of the metabolic biomarker ALT (alanine transami- nase) in the offspring [41]. Studies on animal models have shown similar associations. In rats, a high fat pater- nal diet results in offspring with early onset of impaired y p y p y Obesity is associated with elevated IGF2 circulating levels [44] and increased estrogen levels [45]. Although we did not include IGF2 protein levels in parents or offspring in our current analyses, we earlier showed that hypo- methylation at the IGF2 DMR is associated with higher circulating IGF2 levels in the offspring [25,46]. This asso- ciation was strongest in offspring from obese mothers, independent of race. In brief, a decrease of 5% at the IGF2 DMR methylation corresponded to an increase of at least 10% in serum concentration of IGF2 [46]. Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Offspring of obese fathers may, therefore, demonstrate incomplete methylation. In order to further explore these hypotheses, more research on the epigenetic effects of obesity on human germ cells is needed. Offspring of obese fathers may, therefore, demonstrate incomplete methylation. In order to further explore these hypotheses, more research on the epigenetic effects of obesity on human germ cells is needed. Although our bivariate analysis did not indicate an association between maternal obesity and DNA methyla- tion at the IGF2 DMR in newborns, the regression analy- sis showed that controlling for paternal obesity resulted in an opposite effect, meaning that while paternal obesity was associated with a decrease in methylation, maternal obesity tended to be associated with an increase in methylation, but this was only significant at one CpG site. Using pre-pregnancy BMI instead of obesity in our multivariate analysis strengthened this association. At the H19 DMR, the bivariate analysis showed an increase of 4.1% when mothers were obese (P = 0.01). An increase in DNA methylation by maternal pre-gestational BMI has also been reported earlier in cord blood samples, more particularly at the PPARG promoter [56]. Hypermethyla- tion at the IGF2 or H19 DMR has been associated with loss of imprinting of IGF2, and several disorders. For instance, IGF2 imprinting defects have been implicated in Silver-Russell syndrome [57], Wilms’ tumor [24,58], hepatoblastoma [59] and ovarian cancer [7]. Our regres- sion analyses at the H19 DMR showed that adjusting for potential confounders, including paternal obesity or BMI, diminished this positive association (Table 2, Model 3). We attribute this to the fact that maternal and paternal obesity are closely related and the fact that methylation outcomes for both parental exposures are in the same direction. A larger study is necessary to further explore the potential impact of parental obesity on DNA methy- lation at the H19 DMR. Furthermore, the exposure from oocyte stage till birth to maternal obesity or related life- styles is complex. Hormonal factors that may influence DNA methylation cannot be ruled out. It has been shown that the rat H19 DMR has an estrogen responsive ele- ment, suggesting that estrogen can form a complex with Dnmt1, a DNA methyl transferase, leading to DNA methylation at a normally unmethylated maternal allele [55]. A potential limitation of our study includes the use of cord blood as a marker for the newborn’s epigenetic sta- tus. Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 However, we used isolated leucocytes and IGF2 is a well studied imprinted gene whose germline DMRs should be similarly methylated across all cell types, given the establishment of the epigenetic profile prior to con- ception. IGF2 and H19 DMR methylation profiles were verified in DNA from different cell fractions from umbili- cal cord blood and we found no differences across the cell types [26]. Another possible limitation is proof of paternity, and the reliability of the paternal anthropo- metric data, which were reported by pregnant mothers. However, the questions regarding the anthropometric Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Page 8 of 10 data were detailed and verified for consistency. We do not expect that methylation outcomes are differential with respect to the potential misclassification of expo- sures. Nineteen percent of our population had missing data about the father. The methylation outcomes of these newborns were not included in our final analytical study group. However, we compared methylation outcomes by missing and non-missing characteristics of the fathers and found no differences. We also compared all mea- sured maternal and newborn characteristics in both groups and most characteristics were similarly distribu- ted. We found no significant differences when mothers were single or not, but found significant differences by education and race. Most missing data were among the lower educated and African American mothers. As far as we could test, education was not associated with obesity in either of the parents, and race was only associated with obesity of the mother. It is very likely we missed a number of obese fathers from African Americans. We cannot verify if paternal obesity is equally distributed in all subgroups of missing and non-missing paternal data, and, therefore, selection bias cannot be excluded. How- ever, when reanalyzing our regression models with race included as an independent variable, our results remained the same; only the effect of maternal obesity attenuated in Model 2 (Table 2) at H19 DMR, the b-coef- ficient for maternal obesity became +2.18 (SE = 1.65, P = 0.19) (data not shown). Possibly, race is on the same cau- sal pathway as obesity, regarding the effect on methyla- tion outcomes. As mentioned in the results section, race by itself was not associated with methylation outcomes at neither of the two DMRs studied in this cohort. Never- theless, given our earlier analyses on maternal exposures showed a race dependent effect at the same imprinted loci [53], we further stratified our data by race. The out- comes were in the same direction as the complete analy- tical cohort, but given the smaller numbers, the results represented unstable estimates. In general, the small sam- ple size remains a limitation in our study which may partly explain why our results at the H19 DMR do not reach statistical significance. Nevertheless, the data con- cerning associations between IGF2 DMR methylation and parental BMI or obesity reached sufficient power, especially when studying offspring from fathers with high BMI. Discussion In addition, other studies have shown that small aberrant methylation changes at the IGF2 or H19 DMR is linked to increased expression of IGF2 [6,7,24,25,47], as well as an increased susceptibility to chronic diseases [48-51]. Similar small effects on DNA methylation have also been associated with the use of assisted reproductive technologies [52], the use of psychotropic drugs during pregnancy [53] and smoking [25]. These subtle epigenetic changes have been described as adaptive responses to the environment, while major epigenetic shifts during development would cause more detrimental consequences [25]. Furthermore, explor- ing the CpG sites at the IGF2 and H19 DMRs may repre- sent only a fraction of changes occurring elsewhere in the genome. Environmental factors, among which is diet, have been associated with changes in DNA methylation and may have profound effects on genomic imprinting; accu- mulation of these effects may result in disturbed metabolic homeostasis [54]. Follow-up studies on the anthropo- metric and other developmental factors of the NEST chil- dren are underway to further examine the influence of small changes in DNA methylations at several DMRs on childhood obesity or other adverse consequences. Evidence in animal studies indicates that DNA methyla- tion at the IGF2/H19 locus in sperm might be under tight control of estrogen [55], produced by adipocytes. This sug- gests a mechanism by which increased exposure to estro- gen could lead to inadequate establishment of methylation at the IGF2 DMR in sperm. Alternatively, obesity-related factors may also disrupt functioning of other components of the epigenetic machinery leading to an inability to appropriately establish imprint marks during spermato- genesis, which is ongoing through adult male life [5]. Acknowledgements We thank the participants of the Newborn Epigenetics Study. We also thank the NEST project coordinator Stacy Murray, the data manager Francine Overcash, the research nurse Tammy Bishop, and the laboratory technicians Carole Grenier, Darby Kroyer, Erin Erginer, Cara Davis and Allison Barratt. This work was supported by National Institutes of Health (R21ES014947, R01ES016772, R01DK085173, R25CA126938-01A2), the American Cancer Society (ACS-IRG 83-006), Fulbright, and the Fred and Alice Stanback Foundation. We thank the participants of the Newborn Epigenetics Study. We also thank the NEST project coordinator Stacy Murray, the data manager Francine Overcash, the research nurse Tammy Bishop, and the laboratory technicians Carole Grenier, Darby Kroyer, Erin Erginer, Cara Davis and Allison Barratt. This work was supported by National Institutes of Health (R21ES014947, 16. Painter RC, De Rooij SR, Bossuyt PM, Osmond C, Barker DJ, Bleker OP, Roseboom TJ: A possible link between prenatal exposure to famine and breast cancer: a preliminary study. Am J Hum Biol 2006, 18:853-856. R01ES016772, R01DK085173, R25CA126938-01A2), the American Cancer Society (ACS-IRG 83-006), Fulbright, and the Fred and Alice Stanback Foundation. 17. Dolinoy DC, Weidman JR, Jirtle RL: Epigenetic gene regulation: linking early developmental environment to adult disease. Reprod Toxicol 2007, 23:297-307. 18. Waterland RA, Jirtle RL: Early nutrition, epigenetic changes at transposons and imprinted genes, and enhanced susceptibility to adult chronic diseases. Nutrition 2004, 20:63-68. Competing interests 14. Dolinoy DC, Jirtle RL: Environmental epigenomics in human health and disease. Environ Mol Mutagen 2008, 49:4-8. g The authors declare that they have no competing interests. 15. Painter RC, de Rooij SR, Bossuyt PM, Simmers TA, Osmond C, Barker DJ, Bleker OP, Roseboom TJ: Early onset of coronary artery disease after prenatal exposure to the Dutch famine. Am J Clin Nutr 2006, 84:322-327, quiz 466-327. Abbreviations BMI b d Abbreviations BMI: body mass index; DMRs: differentially methylated regions; ICR: imprinting control region; IGF2: Insulin-like Growth Factor 2; NEST: Newborn Epigenetics Study 9. 9. Steegers-Theunissen RP, Obermann-Borst SA, Kremer D, Lindemans J, Siebel C, Steegers EA, Slagboom PE, Heijmans BT: Periconceptional maternal folic acid use of 400 microg per day is related to increased methylation of the IGF2 gene in the very young child. PLoS One 2009, 4: e7845. Received: 19 April 2012 Accepted: 6 February 2013 Published: 6 February 2013 Authors’ contributions AS developed the hypothesis of this study, designed the analytical strategy, analyzed the data and wrote the manuscript. JS contributed to the analysis and interpretation of the data, and helped to draft the manuscript. AM oversaw participant recruitment in the clinic and contributed to editing the manuscript. FW implemented the statistical analysis. ZH performed the assays. AB contributed to the research discussions and the editing of the manuscript. JK contributed to the logistics of data collection. RJ contributed to the inception of the original NEST research hypothesis. SM is co-principal investigator and oversaw laboratory analysis and processing of the specimens and helped to draft the manuscript. CH is the principle 10. Ollikainen M, Smith KR, Joo EJ, Ng HK, Andronikos R, Novakovic B, Abdul Aziz NK, Carlin JB, Morley R, Saffery R, Craig JM: DNA methylation analysis of multiple tissues from newborn twins reveals both genetic and intrauterine components to variation in the human neonatal epigenome. Hum Mol Genet 2010, 19:4176-4188. 11. Ravelli GP, Stein ZA, Susser MW: Obesity in young men after famine exposure in utero and early infancy. N Engl J Med 1976, 295:349-353. 12. Waterland RA, Jirtle RL: Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 2003, 23:5293-5300. investigator who oversaw the design and conduct of NEST. All authors have read the manuscript and given their final approval of submission for publication. 13. Barker DJ: Intrauterine programming of coronary heart disease and stroke. Acta Paediatr Suppl 1997, 423:178-182, discussion 183. Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Page 9 of 10 References consequence, our preliminary result may be important for public health, especially in regard to the global bur- den of obesity. We hypothesize that the molecular mechanism behind our observation might be a hormo- nal difference between obese and non-obese parents, inducing an incomplete or unstable establishment of methylation at the IGF2 DMR during gametogenesis. As a result, exposures to adverse lifestyle factors or poor/ over-nutrition during spermatogenesis may affect the reprogramming of methylation profiles at imprinted genes. Further research is necessary to confirm this hypothesis. Although our study population is small, this is the first report in humans suggesting that paternal obesity may disrupt the normal establishment of geno- mic imprinting in germ cells. 1. McKay JA, Mathers JC: Diet induced epigenetic changes and their implications for health. 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Author details 1D k C I 1Duke Cancer Institute, Duke University Medical Center 2715, Durham, NC 27710, USA. 2Department of Community and Family Medicine, Duke University Medical Center 104006, Durham, NC 27710, USA. 3Division of Maternal and Fetal Medicine, Department of Obstetrics and Gynecology, Duke University Medical Center, 4022 Hospital South, Durham, NC 27705, USA. 4Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, Duke University Medical Center 91012, Durham, NC 27708, USA. 5Department of Radiation Oncology, Duke University Medical Center 3433, Durham, NC 27710, USA. 6Department of Pediatrics, Duke University Medica Center 3350, Durham, NC 27710, USA. 7Department of Obstetrics and Gynecology, Division of Clinical Epidemiology, Duke University Medical Center 2914, Durham, NC 27710, USA. 19. Levin BE: Metabolic imprinting: critical impact of the perinatal environment on the regulation of energy homeostasis. Philos Trans R Soc Lond B Biol Sci 2006, 361:1107-1121. 20. Boerschmann H, Pfluger M, Henneberger L, Ziegler AG, Hummel S: Prevalence and predictors of overweight and insulin resistance in offspring of mothers with gestational diabetes mellitus. Diabetes Care 2010, 33:1845-1849. 21. Dyer JS, Rosenfeld CR: Metabolic imprinting by prenatal, perinatal, and postnatal overnutrition: a review. Semin Reprod Med 2011, 29:266-276. 22. Tanumihardjo SA, Anderson C, Kaufer-Horwitz M, Bode L, Emenaker NJ, Haqq AM, Satia JA, Silver HJ, Stadler DD: Poverty, obesity, and malnutrition: an international perspective recognizing the paradox. J Am Diet Assoc 2007, 107:1966-1972. Received: 19 April 2012 Accepted: 6 February 2013 Published: 6 February 2013 Page 10 of 10 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Soubry et al. BMC Medicine 2013, 11:29 http://www.biomedcentral.com/1741-7015/11/29 Pre-publication history h bl h The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1741-7015/11/29/prepub doi:10.1186/1741-7015-11-29 Cite this article as: Soubry et al.: Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort. BMC Medicine 2013 11:29. doi:10.1186/1741-7015-11-29 Cite this article as: Soubry et al.: Paternal obesity is associated with IGF2 hypomethylation in newborns: results from a Newborn Epigenetics Study (NEST) cohort. BMC Medicine 2013 11:29. 41. Loomba R, Hwang SJ, O’Donnell CJ, Ellison RC, Vasan RS, D’Agostino RB Sr, Liang TJ, Fox CS: Parental obesity and offspring serum alanine and aspartate aminotransferase levels: the Framingham heart study. Gastroenterology 2008, 134:953-959. 42. Ng SF, Lin RC, Laybutt DR, Barres R, Owens JA, Morris MJ: Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring. Nature 2010, 467:963-966. 43. Dunn GA, Bale TL: Maternal high-fat diet promotes body length increases and insulin insensitivity in second-generation mice. Endocrinology 2009, 150:4999-5009.
https://openalex.org/W3160404001	https://comptes-rendus.academie-sciences.fr/chimie/item/10.5802/crchim.77.pdf	English	null	Energy analysis of a micro-cogeneration unit fed by biogas as a function of pyrolysis operating parameters	Comptes rendus. Chimie	2,021	cc-by	9,130	Antonio Agrifoglioa, Alberto Ficheraa, Antonio Gagliano∗, a and Rosaria Volpe a a Department of Electrical, Electronics and Computer Engineering, University of Catania, viale Andrea Doria 6, 95125 Catania, Italy E-mails: antonio.agrifoglio@outlook.com (A. Agrifoglio), alberto.ﬁchera@unict.it (A. Fichera), antonio.gagliano@unict.it (A. Gagliano), rvolpe@dii.unict.it (R. Volpe) Abstract. In this study, the biomass degradation and the evolution of chemical species during pyrolysis are analysed with the main aim of evaluating the energy performance of a micro-cogeneration unit fed by biogas. The decomposition of the feedstock material is modelled as a two-stage process: ﬁrstly, in the reactor, the biomass is decomposed in a residual solid fraction (char) and a gaseous mixture; then, the condensable gases are divided from permanent gases generating the pyro-oil. The mathematical model proposed in this work has been developed considering the dependence of the pyrolysis process from the temperature and within the interval 500–900 °C. The kinetic of the reactions involved during the pyrolysis was also taken into account. Simulations run in AspenPlus exploiting the R-yield reactor supported by a calculator block. Afterwards, the energy recovery line for the valorisation of the pyro- products has been analysed. The gas fraction obtained at the end of the cycle was ﬁrstly characterized and then used to feed a micro-CHP system. Results are very promising, with great potential in terms of thermal recovery; more than 60% of the initially fed biogas and about 30% power output can be derived. Keywords. Pyrolysis, Simulation, Biomass, Agricultural residue, Micro-CHP, Pyro-gas. Available online 19th May 2021 Volume 24, Special Issue S1 (2021), p. 39-55 Published online: 19 May 2021 Issue date: 4 November 2021 This article is licensed under the Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ This article is licensed under the Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/ Les Comptes Rendus. Chimie sont membres du Centre Mersenne pour l’édition scientiﬁque ouverte www.centre-mersenne.org e-ISSN : 1878-1543 Les Comptes Rendus. Chimie sont membres du Centre Mersenne pour l’édition scientiﬁque ouverte www.centre-mersenne.org e-ISSN : 1878-1543 Comptes Rendus Chimie 2021, 24, nS1, p. 39-55 https://doi.org/10.5802/crchim.77 Comptes Rendus Chimie 2021, 24, nS1, p. 39-55 https://doi.org/10.5802/crchim.77 Sustainable Biomass Resources for Environmental, Agronomic, Biomaterials and Energy Applications 2 / Ressources de biomasse durables pour des applications environnementales agronomiques, de biomatériaux et énergétiques 2 ∗Corresponding author. https://comptes-rendus.academie-sciences.fr/chimie/ Sustainable Biomass Resources for Environmental, Agronomic, Biomaterials and Energy Applications 2 / Ressources de biomasse durables pour des applications environnementales, agronomiques, de biomatériaux et énergétiques 2 1. Introduction Diﬀerently from the previous experimentally driven contributions, Di Blasi devel- oped a mathematical model to couple the eﬀects de- riving from heat, momentum and mass transfer phe- nomena with the thermal degradation of wood [12]. A numerical study is also presented in the work of Babu and Chaurasia [13], who investigated the opti- mum set of parameters (temperature, concentration, time and heating rates) for the pyrolysis of biomass. In Table 1, a synthetic framework for prediction- based models tackled from the aforementioned lit- erature is oﬀered. Each paper has been classiﬁed as a function of the operative temperature range, the feedstock biomass, modelling or numerical ap- proaches vs. experimental investigation and the kind of analyses. As emerged from the literature review, several ex- perimental studies and models have been proposed by researchers with respect to the chemical and phys- ical behaviours of diﬀerent types of feedstock mate- rial during pyrolysis. However, due to the intrinsic complexity of the chemical evolution of biomass, it may be useful to develop numerical simulations able to oﬀer a sim- pliﬁed but reliable description of the pyro-products yields and their chemical decomposition. In this per- spective and drawing lessons from the existing litera- ture, a novel simulation model for biomass pyrolysis is presented in this study. The proposed model includes an accurate charac- terization of the initial feedstock material and pre- dicts the yield and composition of pyrolysis prod- ucts in order to estimate to what extent the produced pyro-gas can be exploited for energetic purposes, i.e. to recover thermal energy to support plant opera- tions. In comparison to existing numerical pyrolysis models which provide a simpliﬁed composition of the produced pyrogas, the presented model proposes a very detailed analysis of the chemical species origi- nating from the pyrolysis process. Thus, in general terms, biomass pyrolysis can be studied considering the initial feedstock composi- tion [14], reaction pathways [4,15] and ﬁnal prod- ucts composition [16]. Apart from the chemical view- point, pyrolysis has been extensively studied either as a stand-alone process or as an integration to gasiﬁca- tion processes [17]. In particular, simulations can be targeted to speciﬁc pyrolysis plants [18] and includ- ing sensitivity analysis [19] or can be directed to in- vestigate dedicated energy recovery lines [20,21], but rarely investigations in the literature include both as- pects. 1. Introduction at which the pyrolytic process occurs as well as on the chemical reactions taking place during the inter- mediate decomposition of the feedstock material [2]. With respect to the biochemical constituents, cellu- lose, hemicellulose and lignin have the highest poly- meric distribution in plants and, generally, in plant- derived materials. Therefore, the study of their ther- mal degradation is fundamental to gain awareness on the feedstock chemical composition, on its ther- Pyrolysis consists of the thermal decomposition of carbonaceous materials (generally wood or agricul- tural residues) into a solid, liquid and gaseous frac- tion in absence of oxygen [1]. Products originating from pyrolysis strongly depend on the temperature ISSN (electronic) : 1878-1543 https://comptes-rendus.academie-sciences.fr/chimie/ 40 Antonio Agrifoglio et al. mal stability and the decomposition kinetics of the pyrolysis process. process of biomass (including the chemical evolu- tion of secondary reactions) and the energy perfor- mance evaluation of a micro-cogeneration unit cou- pled with an energy recovery line. As studied by Ranzi et al. [3], a way to deal with the chemistry of pyrolysis consists in the character- ization of the initial biomass and both primary and secondary gasiﬁcation reactions. In their paper, the authors developed a model to predict the gas com- position deriving from biomass gasiﬁers particularly focusing on primary constituents (cellulose, hemi- cellulose and lignin) and their degradation. In their subsequent work, they derived the yield of gas, tar and solid residue [4]. A similar contribution can be recognized in the study of Liden et al. [5] focusing on the kinetic prediction model for the liquid frac- tion from fast pyrolysis. On the same issue, but fo- cussing on slow pyrolysis, Hu et al. [6] compared dif- ferent kinetic models on the ground of data derived from thermogravimetric analyses (TGA). Carrier et al. [7] based their research on the application of TGA to determine the chemical structure of biomass feedstock. The study of the thermal behaviour of biomass by thermogravimetric analysis is also tack- led by Zhou et al. [8] and by Zhang et al. [9]. TGA data are also used by Huang et al. [10] to predict chemi- cal kinetics. Lee et al. [11] developed a methodology based on experimental analyses in order to derive the chemical composition of gaseous products orig- inating from pyrolysis. C. R. Chimie — 2021, 24, nS1, 39-55 1. Introduction To ﬁll this gap, in this paper, simulations aim at increasing awareness on both the decomposition This high granularity is a strategic detail if plan- ning to exploit the produced biogas to fed mechani- cal engines, for which the exact knowledge about the chemical composition is necessary. The relationships between biomass and its primary constituents are used to accurately correlate the degradation of the feedstock material with the evolution of the chemical species originating from pyrolysis. As a further added value, the decomposition model takes into account both the dependence of degradation from tempera- ture and the kinetics of reactions involved during the process. The model provides information not only on the evolution of the main pyro-products as a function 41 Antonio Agrifoglio et al. Table 1. A synthetic summary of the literature review Paper Temperature range Feedstock biomass Modelling approach/ experimental investigation Analyses Ranzi et al. [3] 100–1000 °C Softwood Hardwood Experimental Gas composition prediction Liden et al. [5] 400–700 °C Wood Experimental Organic liquid yield prediction Hu et al. [6] 150–650 °C Cellulose Thermogravimetric experiments Biomass decomposition Di Blasi [12] 100–1000 °C Wood — Heat transfer and secondary reactions Blanco Lopez et al. [16] 100–700 °C Olive stones Experimental Gas mixture composition Carrier et al. [7] 100–700 °C Wood and fern Thermogravimetric experiments Fractionation of biomass Benanti et al. [19] 100–600 °C Olive pits CHEMCAD model and experimental measurements Simulation of char production vs. experimental data Ducom et al. [20] 100–900 °C Olive residues Experimental Energy recovery Table 1. A synthetic summary of the literature review Energy recovery of the temperature but also with respect to the ther- mal degradation of chemical species. 300 °C, with mass loss maximum peak around 340–360 °C. At higher temperature, typically over 400 °C, almost all cellulose has been pyrolysed leav- ing a solid residue of circa 7%. Hemicellulose de- composes easier than cellulose, usually between 200–300 °C, with mass loss peak around 270 °C. At the end of the pyrolysis process, however, a residue of circa 20% of hemicellulose can still be recognized. Lignin decomposition occurs within a large temper- ature range (from 100 °C to 900 °C), presenting lower conversion rates and lower mass losses compared to cellulose and hemicellulose; in this case, the solid residue from pyrolysis is around 45% of the initial feedstock. The paper is structured as follows. 1. Introduction Section 2 presents the materials and methods with a focus on the feedstock composition, the modelling of the de- composition process and the characterization of ﬁnal products. In addition, the simulation scheme and the modelling of the implemented energy recovery line are explained. Section 3 introduces the case study and discusses the main results of the simulations for both the primary and secondary reactions and the energy performances of the micro-cogeneration unit and the thermal recovery line. Going into the detail of intermediate compounds from biomass pyrolysis, at the increase of the tem- perature, cellulose separates into water and car- bon from the initial starting matrix and results in an intermediate compound called “active cellulose”, CELL-A [3,23]. The further degradation of CELL-A results in the formation of Levoglucosan, a charac- teristic compound deriving from cellulose pyrolysis, and a mixture of other minor components. Hemicel- lulose (HCE) ﬁrstly decomposes into the two main intermediate compounds: HCE1 and HCE2. Three C. R. Chimie — 2021, 24, nS1, 39-55 2.1. Preliminary consideration about the feed- stock composition 2.1. Preliminary consideration about the feed- stock composition Biomass properties are usually determined by tak- ing into consideration the biochemical and ele- mentary composition and the energy content. With respect to the biochemical constituents, as under- lined by Chen et al. [15] and Yang et al. [22], the de- composition process for cellulose begins around C. R. Chimie — 2021, 24, nS1, 39-55 Antonio Agrifoglio et al. 42 Figure 1. Biomass decomposition scheme. diﬀerent sub-reactions, one of them yielding the Xy- losan, characterise the HCE1 decomposition, whilst HCE2 decomposed in a mixture of minor compo- nents [3,23]. The thermal degradation of lignin is more complex if compared to cellulose and hemicel- lulose. Lignin decomposition yields three main in- termediate products, each of them characterized by the dominance of either carbon (C), hydrogen (H) or oxygen (O) and indicated, respectively, as LIG–C, LIG–H and LIG–O. The LIG–C decomposes in LIG–CC and a mixture of common compounds and from LIG– CC Coumaryl is obtained. Going forward, LIG–H and LIG–O interact to obtain LIG–OH from which Sina- paldehyde originates. The pyrolysis of lignin is also responsible for the production of Phenol. Figure 1. Biomass decomposition scheme. The initial feedstock decomposes into solid char and a gaseous fraction. The solid fraction (char) is mainly constituted by carbon and contains volatiles and ash; the gaseous fraction includes both condens- able and non-condensable gases. Due to cooling and cleaning gas systems, the condensable gaseous frac- tion undergoes further secondary reactions yielding a liquid fraction (composed of water and pyrolytic oil) and a permanent gaseous fraction. Thus, in com- pliance with the scheme of Figure 1, the mass balance equation of the entire decomposition process can be written as: To build a reliable feedstock, it is fundamental to consider the elemental composition of biomass along with its biochemical characterization. In this study, the elemental composition is derived from previous studies available in literature [16,24,25] and free online databases [26]. Thus, the prelimi- nary biomass structure was obtained by rearranging the information obtained from elemental compo- sition and the stoichiometric balances of complete biomass thermo-chemical decomposition derived from the literature [3]. From this obtained feedstock, the atomic ratios H/C and O/C ratios were calcu- lated. 2.1. Preliminary consideration about the feed- stock composition (2.2) m f = mc +mg1 = mc +ml +mg2 (2.2) Being m f the mass of the inlet feedstock in the reac- tor, mc the mass of the solid char and mg1 the mass of the ﬁrst-reaction gaseous products (including both condensable and non-condensable gases). This latter is constituted by the mass of the liquid products ml and the non-condensable gaseous fraction mg2. To ensure the accuracy of simulations, the kinetic of the chemical decompositions should be repro- duced taking into consideration all factors mostly aﬀecting the pyrolysis. The mass changes occurring during the reactions can be calculated from the TG- Analysis through the calculation of the conversion rate α, expressed as: The energy content of biomass is described by the higher heating value (HHV) calculated using the em- pirical correlation developed by Ebeling and Jenk- ins [27], reported in (2.1): HHV = −0.763+0.301[C]+0.525[H]+0.064[O] (2.1) This correlation is valid for any biomass species and correlates the percentages of C, H and O derived from the ultimate analysis. α = mi −m mi −m f (2.3) (2.3) in which mi and m f are the initial and ﬁnal mass respectively, and m the current mass. The evolution of the species involved during the pyrolysis can be written as: 2.2. Modelling of the decomposition process The decomposition of biomass can be modelled with respect to just the primary or to both the primary and secondary reactions [12]. The initial feedstock de- composes into solid, liquid and gaseous products; in particular, primary reactions produce intermediates that evolve in the ﬁnal products after the secondary reactions [28]. In this work, a two-steps decom- position scheme has been adopted, as reported in Figure 1. dα dt = k · f (T ) (2.4) (2.4) The derivative of the conversion rate α in (2.4) de- pends on two factors: the coeﬃcient k, expressing the heating rate and necessary to describe the kinet- ics of the reaction, and the polynomial function f (T ) correlating the species in the compound with the in- crease of temperature. C. R. Chimie — 2021, 24, nS1, 39-55 43 Antonio Agrifoglio et al. Figure 2. Software simulation logic. Figure 2. Software simulation logic. The kinetic law of any chemical reaction is gov- erned by the Arrhenius equation as expressed in (2.5): correlations. These are already available in the soft- ware library or are developed by users and fed to the calculator block tools. This simulation logic is sum- marized in Figure 2. k = k0 ·e−Ea RT (2.5) (2.5) where the coeﬃcient k is the constant rate and de- scribes the degree of decomposition, k0 is a pre- exponential factor, Ea is the activation energy, R = 8.314 J/(mol·K) is the universal gas constant and T the absolute temperature. The simulation of biomass decomposition is a complex issue, for which dedicated models are not available within software packages [31,32]. A way to overcome this diﬃculty is to build a user-deﬁned ma- terial, usually referred to as a nonconventional com- ponent, in order to set the composition and char- acteristics of the feedstock material [29]. Several ex- amples of implemented operation blocks simulat- ing thermochemical conversion processes have been adopted from the current literature. For instance, Benanti et al. simulated a plug ﬂow reactor to study the correlation between temperature and composi- tion of the ﬁnal products obtained by slow pyroly- sis [19]. In their study, they used the decomposition reactions of biomass to obtain the input kinetic pa- rameters. Fonseca et al. chose a Yield Reactor block for simulating wheat straw as nonconventional ma- terial and calculated properties and heat capacity of the obtained products from fast pyrolysis [33]. 2.3. Simulation scheme The understanding of kinetics reactions underly- ing the pyrolysis process is a multi-faceted prob- lem. To build a reliable simulation model, the chem- ical species involved during pyrolysis as well as their physical and chemical characteristics should be properly speciﬁed as input parameters within any simulation environment. Indeed, the main idea be- hind the simulations consists of the right choice of a layout ensuring the interaction between com- pounds and unit blocks [29,30]. Calculation of mass and energy balances, thermodynamic equilibrium, rate equations, mass-ﬂow rate, temperature, pressure are based on mathematical models and empirical 2.2. Modelling of the decomposition process The polynomial function f (T ) can be determined through the percentages of each species for a temper- ature range of 773 < T < 1173 K: f (T ) = a +bT +cT 2 (2.6) (2.6) The coeﬃcients a, b and c were determined through interpolation from the data available in literature. C. R. Chimie — 2021, 24, nS1, 39-55 2.4. Characterization of ﬁnal products The heat capacity of the gas mixture can be calcu- lated according to (2.8): cp,g = xX n=1 yn ·cp,n (2.7) (2.7) Being yn and cp,n the mass fraction and heat ca- pacity of each chemical species and x the number of chemical species. In detail, the heat capacities of C. R. Chimie — 2021, 24, nS1, 39-55 44 Antonio Agrifoglio et al. each component have been calculated according to the NASA polynomial method [34,35]: can be calculated. However, considering complete combustion and high performances for the engine, the air/fuel equivalence ratio λ is used; in this case, the actual air/fuel ratio A/F is obtained by taking into account the excess of air: cp,n R = a1 + a2T + a3T 2 + a4T 3 + a5T 4 (2.8) (2.8) where the coeﬃcients a1−5 are speciﬁcally deﬁned for each compound and are tabulated in litera- ture [34,35]. Final products diﬀer for their physical states and their chemical compositions. Therefore, to estimate their higher heating values (HHV), it is nec- essary to implement purpose-built methods. Regard- ing gaseous compounds and depending on the pres- ence of oxygen, the HHV can be calculated according to (9) and (10), as suggested by Demirbas et al. [36]: A/F = λ· A/Fstoich (2.14) (2.14) The recycling system is schematically represented in Figure 3. The recycling system is schematically represented in Figure 3. In accordance with the speciﬁcation of the CHP system, thermal energy from cooling water and ex- haust gas can be recovered by a heat exchanger and recirculated [39]. The thermal energy from cooling water Qcw is calculated as: HHV = 0.303[C]+1.423[H] (2.9) HHV = 0.305[C]+1.423[H]−0.154[O] (2.10) The HHV for oil can be expressed by the for- mula [37]: HHV = 0.303[C]+1.423[H] (2.9) HHV = 0.305[C]+1.423[H]−0.154[O] (2.10) The HHV for oil can be expressed by the for- mula [37]: HHV = 0.303[C]+1.423[H] Qcw = mcw ·cp,cw ·∆Tcw (2.15) (2.15) HHV = 0.305[C]+1.423[H]−0.154[O] (2.10) The HHV for oil can be expressed by the for- mula [37]: In (2.15), mcw and ∆Tcw are the mass of the cooling water and the temperature variation, both derived from the datasheet of the equipment; cp,cw is the heat capacity of water. 2.4. Characterization of ﬁnal products HHV = 0.325[C]+0.944[H]−0.105[O] (2.11) HHV = 0.325[C]+0.944[H]−0.105[O] (2.11) (2.11) Eventually, for both products, the lower heating value (LHV) can be expressed using (2.12), as recom- mended in [1]: Similarly thermal energy Qex is deﬁned as: Qex = mex ·cp,ex ·∆Tex (2.16) (2.16) Being mex = mair + msyn the total mass of the ex- haust gas (considering the mass of air and the mass of syngas, respectively), ∆Tex = Ti,ex−To,ex the tempera- ture variations and cp,ex the heat capacity of exhaust. LHV = HHV−hv µ9[H] 100 + M 100 ¶ (2.12) where hv is the latent heat of vaporization and M the moisture content of gas or pyro-oil. p, p y The mechanical power Pm generated by the sys- tem can be calculated as: 2.5. Recovering Pm = Qsyn −Qcw −Qex −Ql (2.17) (2.17) One way for valorising the gas obtained from pyrol- ysis is to feed an engine connected to an electrical generator for the simultaneous production of elec- trical and thermal energy. The heat released during the combustion process is recovered from the cooling water and exhaust gases and redirected to the main plant at the service of drying and/or pyrolysis stages. Where Qsyn = m ·LHVsyn is the heat generated by the syngas combustion and Ql the heat losses due to the combustion chamber, mechanical friction and lubri- cation. Finally, considering the alternator eﬃciency ηgen, the produced electrical power Pel is: Pel = Pm ·ηgen (2.18) (2.18) The theoretical quantity of oxygen mO2 necessary to complete the combustion of a fuel can be ex- pressed as [38]: C. R. Chimie — 2021, 24, nS1, 39-55 C. R. Chimie — 2021, 24, nS1, 39-55 3. Case study and results In this study, olive pits have been chosen as feedstock material. Their biochemical and elemental compo- sitions have been determined by averaging data and experimental measures available from previous works of the authors [24,40], other current litera- ture [16] and online databases [26]. The ﬁnal feed- stock has been modelled as a non-conventional solid named “Biomass”, which biochemical and elemental compositions, molecular weight, H/C and O/C ratios and caloriﬁc value have been reported in Table 2. mO2 = YC · ·MO2 MC ¸ + YH 4 · ·MO2 MH ¸ +YS · ·MO2 MS ¸ −YO (2.13) mO2 = YC · ·MO2 MC ¸ + YH 4 · ·MO2 MH ¸ +YS · ·MO2 MS ¸ −YO (2.13) In this equation, Mi is the molecular mass of each compound i, being i = O2, CO2 (kgi/kmol), M j the molecular mass of element j, being j = C, H,S,O (kgj /kmol) and, Yj the mass fraction of ele- ment j, with j = C,H,S,O (kgj /kgfuel). j From these data, and knowing the oxygen percent- age in air, the stoichiometric air/fuel ratio A/Fstoich C. R. Chimie — 2021, 24, nS1, 39-55 45 Antonio Agrifoglio et al. Figure 3. Cogeneration system cycle. Table 2. Biochemical distribution in the ﬁnal feedstock Component [C] (wt%) [H] (wt%) [O] (wt%) MW (g/mol) H/C (—) O/C (—) Cell (wt%) HC (wt%) Lig (wt%) HHV (MJ/kg) Biomass 49.43 6.03 44.54 4130.76 1.453 0.67 31.40 41.58 26.02 20.13 Simulations run considering 30 kg/h of feedstock 24,41,42]. The coeﬃcients a, b and c used to perform Figure 3. Cogeneration system cycle. Figure 3. Cogeneration system cycle. Table 2. Biochemical distribution in the ﬁnal feedstock Table 2. Biochemical distribution in the ﬁnal feedstock Table 2. Biochemical distribution in the ﬁnal feedstock Component [C] (wt%) [H] (wt%) [O] (wt%) MW (g/mol) H/C (—) O/C (—) Cell (wt%) HC (wt%) Lig (wt%) HHV (MJ/kg) Biomass 49.43 6.03 44.54 4130.76 1.453 0.67 31.40 41.58 26.02 20.13 Simulations run considering 30 kg/h of feedstock material with 15% of moisture content. The calcula- tor block processes all components entering the re- actor: in this process, the temperature is an import variable, whilst the twenty-one chemical species in- volved during the thermochemical conversion repre- sent export variables. During the conversion, the evo- lution of each compound is analysed within the tem- perature range 500–900 °C. It is worth pointing that, in this study, the high granularity of the considered chemical species enhance the validity of the pro- posed simulation model and ensures a more com- prehensive characterization from the chemical evo- lution viewpoint. Th di t ib ti l i bt i d b bi i 24,41,42]. The coeﬃcients a, b and c used to perform the evolution of the conversion rates of each com- pound are obtained by comparison among empirical and literature results and are listed in Table 3. Simulations were carried out using ASPEN PLUS [29]; the plant design and the ﬂow pro- cess used to perform the pyrolysis are reported in Figure 4. The initial feedstock passes through the inlet (S1). The dryer (B1) removes the moisture from the feedstock and ejects it at (S3). Afterwards, both the daf (dry and ash-free) and pre-heated materials are fed through (S2) to the Yield Reactor (B2). The thermochemical conversion of biomass is simu- lated by coupling the reactor with a calculator block. More precisely, the chosen reactor does not require exact information on the stoichiometry or kinetic of the material; users just need to specify the pro- duction yields of the involved chemical species. It is worth noting that, being these speciﬁcations independent from the temperature, the calculator block can be usefully implemented to deal with the lution viewpoint. The distribution law is obtained by combining the Arrhenius equation in (2.5) and the polynomial function of the temperature expressed in (2.6). The derived equation for the mass mx of the x compound is reported in (3.1): mx = k · f (T ) = k0 ·e ¡ −Ea RT ¢ · a +bT +cT 2 (3.1) (3.1) Data derived from empirical studies and literature have been adopted to develop the simulation [14,18, C. R. Table 2. Biochemical distribution in the ﬁnal feedstock Chimie — 2021, 24, nS1, 39-55 46 Antonio Agrifoglio et al. Table 3. Coeﬃcients for yield distribution Compound a b c C2H4O2 HAA 7.12 −0.01273 5.76×10−6 C2H2O2 GLYOXAL 0.979 0.00115 −1.62×10−6 C2H4O ACETA −13.98 0.031 −1.6×10−5 C6H6O3 HMFU −30.65 0.0688 −3.7×10−5 C3H6O PROPILEN 1.61 −0.00387 2.32×10−6 CO2 C-DIOX 6.678 −0.0105 4.84×10−6 CO C-MON −14.48 0.03041 −1.4×10−5 CH4 METHANE 5.204 −0.0118 7.00×10−6 H2 HYDROG −24.7 0.0482 −2.1×10−5 H2O WATER 10.86 −0.01615 7.53×10−6 CH2O2 FORMIC AC −45.05 0.107 −5.8×10−5 C CHAR 12.91 −0.02248 1.02×10−5 C6H10O5 LVG −49191 109.97 −0.05743 CH2O FORMALD −7.91 0.017824 −9.44×10−6 C2H6O ETHANOL −4.13613 0.007301 −2.91×10−6 CH3OH METHANOL −3.09 0.00718 −3.87×10−6 C2H4 ETHYLENE 4.14 −0.01 6.03×10−6 C5H8O4 XYLOSAN −4345 8.31 −0.00179 C9H10O2 COUMARYL −27.89 0.0374 9.93×10−6 C6H6O PHENOL −27.7 0.0273 3.83×10−5 C11H12O4 FE2MACR 90610 −215.7 0.1285 Table 3. Coeﬃcients for yield distribution (S8), i.e. at the inlet and outlet of the scrubber, and cp,w (kJ/kg·K) the heat capacity of water. At the nu- merator, ˙mg and Ti,g are the mass ﬂow rate and the temperature of the gas at the inlet of the line (S7). These values are deﬁned in the calculator block as imported variables. formation of chemical species [43]. Outlet products from the reactor are directed in the cyclone (B3) for the gas–solid separation. The gas fraction is ﬁrstly cooled in a heat exchanger (B4) and then directed to the cleaning/washing section, simulated as a dis- tillation column (B5). A second calculator is used to set the amount of water needed to enable the condensation process in the scrubber, also taking into consideration that the gas mixture leaving the reactor depends on the temperature. A water ﬂow is then injected from (S8) to decrease the tempera- ture and to separate the condensable products from the permanent gas. The water ﬂow rate ˙mw (kg/h) is an export-variable of the H2O calculator and is formulated as: formation of chemical species [43]. Outlet products from the reactor are directed in the cyclone (B3) for the gas–solid separation. The gas fraction is ﬁrstly cooled in a heat exchanger (B4) and then directed to the cleaning/washing section, simulated as a dis- tillation column (B5). A second calculator is used to set the amount of water needed to enable the condensation process in the scrubber, also taking into consideration that the gas mixture leaving the reactor depends on the temperature. C. R. Chimie — 2021, 24, nS1, 39-55 Table 2. Biochemical distribution in the ﬁnal feedstock A water ﬂow is then injected from (S8) to decrease the tempera- ture and to separate the condensable products from the permanent gas. The water ﬂow rate ˙mw (kg/h) is an export-variable of the H2O calculator and is formulated as: The yields of char, liquid and gaseous fractions as a function of the temperature are reported in Fig- ure 5, whilst the evolution of the chemical species in the range 500–900 °C is illustrated in Figure 6. Char production has a decreasing trend up to 750 °C beyond which it becomes almost constant. Liquid production decreases slowly and continu- ously with temperature; this fraction needs some re- ﬁnery stage to yield chemicals and biofuels. C y Liquid production decreases slowly and continu- ously with temperature; this fraction needs some re- ﬁnery stage to yield chemicals and biofuels. Gas production shows a rapid increase up to 850 °C and then becomes stable at 900 °C. As said, the gas obtained from this process can be used to feed an engine for the cogeneration of electrical and thermal energy. ˙mw = ˙mg cp,g (Tf ,g −Ti,g ) cp,w(Tf ,w −Ti,w) (3.2) (3.2) In this equation, Ti,w and Tf ,w (°C) are, respec- tively, the initial and ﬁnal temperature of water in In this equation, Ti,w and Tf ,w (°C) are, respec- tively, the initial and ﬁnal temperature of water in C. R. Chimie — 2021, 24, nS1, 39-55 Antonio Agrifoglio et al. 47 Figure 4. Simulation plant design and ﬂow process. Figure 5. Yields of the solid, liquid and gaseous fractions at the increase of the temperature. The yield of the three main pyrolysis products ity or concavity of the curve: indeed in (3 3) as a re 47 Antonio Agrifoglio et al. Figure 4. Simulation plant design and ﬂow process. Figure 4. Simulation plant design and ﬂow process. Figure 4. Simulation plant design and ﬂow process. Figure 5. Yields of the solid, liquid and gaseous fractions at the increase of the temperature. Figure 5. Yields of the solid, liquid and gaseous fractions at the increase of the temperature. The yield of the three main pyrolysis products as a function of the temperature can be described by a polynomial or a linear interpolation. Interpo- lations provide a simple but eﬀective way to esti- mate the analytical expression of any process. As can be seen from Figure 5, char and gas produc- tions are well characterized by a quadratic function y = aT 2 +bT +c, being respectively: ity or concavity of the curve: indeed, in (3.3), as a re- sult of the positive sign of the second-order term, the curve of the char yield is convex, whilst on the other case, in (3.4), the curve of the gas yield is concave. Oil yield is better described by a simple linear in- terpolation y = mT +c, such as: yoil = −0.018T +0.2435 (3.5) (3.5) yoil = −0.018T +0.2435 ychar = 0.0052T 2 −0.0776T +0.4145 (3.3) ygas = −0.005T 2 +0.0933T +0.3462 (3.4) The analytical expression in (3.5) implies a con- stant decreasing trend of oil production (also con- The sign of the term a indicates either the convex- C. R. Chimie — 2021, 24, nS1, 39-55 48 Antonio Agrifoglio et al. ﬁrmed by the negative sign of the slope m) at increas- ing the temperature. around 750–800 °C. The increase of H2 combined with the increase of CO and CH4 and a consequent drop of CO2 signiﬁcantly changes the composition and properties of pyro-gas. The water content is ap- proximately constant during the considered interval. Cellulose, hemicellulose and lignin contribute to the production of methane CH4, which becomes partic- ularly evident at higher temperatures, i.e. in corre- spondence with the higher production of pyro-gas. A common way to evaluate the eﬀectiveness of the derived analytical expressions consists of calculating the coeﬃcient of determination R2. Values nearby the unit implies a good/optimal prediction capability of the model, i.e. a more than acceptable goodness of ﬁt. From this perspective, as can be read in Figure 5, the derived analytical interpolations (both the qua- dratic for char and gas and the linear one character- izing the oil) can be considered satisfactory. Figure 4. Simulation plant design and ﬂow process. Going into the detail of the main biochemical components of biomass, during pyrolysis, cellulose degrades into water and carbon and, as a secondary reaction, in active cellulose CELL-A from which Levoglucosan LVG and other minor components derive. As can be seen, the percentage of CELL-A converted into LVG increases at higher temperatures, reaching a maximum of around 650–700 °C. Be- yond LVG, cellulose thermal degradation produces other volatiles such as Hydroxyacetaldehyde HAA and Glyoxal, two of the main products deriving from pyrolysis of carbohydrates in biomass and directly aﬀecting the formation of char (coherently with the fact that lower temperatures favour charring reac- tions and with the trend of HAA). At higher tempera- tures, typically beyond 600 °C, HAA decomposes into methane CH4, carbon monoxide CO, carbon dioxide CO2, Hydroxymethylfulfural HMFU, formaldehyde CH2O, ketene C2H2O and acetaldehyde C2H4O. As said, the coeﬃcient of determination is a good indicator to conﬁrm the eﬀectiveness of a given an- alytical law; however, to be more accurate, it could be helpful to include the evaluation of the coeﬃcient of correlation r in the analysis as it allows measur- ing the strength of the analytical relation between the two variables concerning the simulated dataset. The coeﬃcient of correlation r can be either pos- itive or negative: if one of the considered variable increases (as in the case of the temperature in the simulation model), the other variable increases (pos- itive correlation) or decreases (negative correlation), as well. It can be calculated as the ratio between the covariance of two variables and the product of their standard deviations. In this case, the coeﬃcients of linear correlation are rchar = −0.9065, roil = −0.998 and rgas = 0.9675. The decomposition scheme for hemicellulose proposed by Ranzi et al. [4] yields the two main acti- vated hemicellulose, HCE1 and HCE2. The ﬁrst com- pound, HCE1, decomposes into Xylosan and ethanol C2H5OH, whose evolutions display a slight increase at increasing the temperature. In minor percent- ages, other components deriving from the hemicel- lulose sub-components are methane CH4, formalde- hyde CH2O and methanol CH3OH. The evolution of these last two compounds, i.e. formaldehyde and methanol, almost imitates the trends of the chemical species originating from cellulose, such as LVG and HMFU. C. R. Chimie — 2021, 24, nS1, 39-55 Figure 4. Simulation plant design and ﬂow process. In this sense, from the qualitative viewpoint, the pyrolysis of cellulose and hemicellulose is quite similar and the decomposition schemes for both components with respect to the temperature can be easily compared. It is worth pointing that the coeﬃcient of lin- ear correlation implies some approximations for char and gas, both characterized by a quadratic expres- sion, remaining however highly descriptive for the scope of this study. As can be noted, the coeﬃcient of linear correlation is negative in the case of char and oil, highlighting that the increase of the temperature produces a decrease in the yield of both components. Vice versa for the gas, for which the direction of the relationship is positive, i.e. higher temperatures per- mit to achieve a higher amount of pyro-gas. As regards the prediction of the chemical species, Figure 6 depicts the evolution of the main com- pounds deriving from the pyrolysis of cellulose, hemicellulose and lignin at increasing temperature. The chemical species with higher percentages are re- ported in Figure 6(a). A drop of carbon (C) can be ob- served between 450 °C and 700 °C probably due to the decrease of the char production in this temperature range. Similarly, CO2 decreases, whilst H2 and CO rapidly increase becoming the two main components Finally, following the scheme of Ranzi et al. [3], lignin decomposes into LIG–C, LIG–H and LIG– O. The ﬁrst sub-component, LIG–C, mainly de- composes into LIG–CC, whose evolution produces Antonio Agrifoglio et al. 49 Figure 6. Evolution of the chemical species. Figure 6. Evolution of the chemical species. Coumaryl, phenol C6H6O and, in smaller quanti- ties, methane CH4, carbon monoxide CO and car- bon dioxide CO2. The other two sub-components, LIG–H and LIG–O, interact to obtain LIG–OH that decomposes into Sinapaldehyde FE2MACR. 3.5 kg/h of the initial feedstock. The remaining frac- tion, i.e. 26.5 kg/h, corresponds to the gas mixture ex- iting from the cyclone and moving towards the scrub- ber. In this section, Equations (2.7) and (3.2) are used into the second calculator block to deﬁne the heat capacity of the gas mixture and the water mass ﬂow rate needed to cool the gas mixture and to separate the condensable products from permanent gas. In this speciﬁc case, these calculations give as results cp,g = 3.498 kJ/kg·K and ˙mw = 73.5 kg/h. C. R. Chimie — 2021, 24, nS1, 39-55 3.1. Energy recovery line To optimize the process, an energy recovery line con- sisting of a micro-CHP system with output power up to 100 kWe has been taken into account [44]. Calculations are based on the REC2-40G system of Enerblu Industry [45], working with an electrical output of 43 kWe in full-load and 22 kWe in half-load. The working temperature of 800 °C is chosen to in- vestigate to what extent the heat generated from the combustion of the gaseous fraction in a CHP system can be recovered to feed the process. Outlet products from the reactor (S4), as obtained from the ﬁrst cal- culator block using (3.1), are reported in Table 4. Figure 7 reports the products distribution at the end of the cycle. As can be seen, the yield of permanent gas is signiﬁcantly higher than the other two pyrolysis products and has a production rate of circa 75%; char and oil have comparable yields, around the 12%. Tables 5, 6 and 7 summarize the composition of the solid, liquid and gaseous fraction, respectively. Here, again, the high granularity of the chemical species can be considered as a beneﬁt for the model, giving that such level of detail also leads to more These products enter the cyclone (B3) that sepa- rates about the 97.47% of the produced char, i.e. circa C. R. Chimie — 2021, 24, nS1, 39-55 50 Antonio Agrifoglio et al. Table 4. Products at the reactor outlet Compound kmol/h mol% kg/h wt% CO2 0.097393 2.99871 4.286245 14.28748 HCOOH 0.005385 0.1658 0.247844 0.826146 HAA 0.004189 0.128977 0.251558 0.838526 H2 2.16926 66.79113 4.372967 14.57656 CH4 0.068515 2.109562 1.09917 3.6639 HMFU 0.002608 0.08029 0.328859 1.096196 LVG 6.07×10−3 0.187036 0.984952 3.283173 C2H5OH 0.001879 0.057842 0.086546 0.288487 C2H4 0.009743 0.299986 0.273329 0.911095 Coumaryl 0.003285 0.101144 0.493327 1.644425 FE2MACR 5.61×10−4 0.017274 1.17×10−1 0.38937 Phenol 0.007979 0.245676 0.75094 2.503134 Xylosan 1.03×10−3 0.031813 1.37×10−1 0.455022 CH3OH 0.002612 0.08041 0.083681 0.278936 CH2O 0.026043 0.80185 0.781966 2.606554 C3H6O 0.004576 0.140909 2.66×10−1 0.886008 C2H4O 0.006188 0.190543 0.272622 0.908741 Glyoxal 0.002871 0.088397 0.166622 0.555405 CO 0.187901 5.785441 5.263186 17.54395 Char 0.297766 9.168156 3.576465 11.92155 H2O 0.341965 10.52905 6.160602 20.53534 Total ﬂow 3.247826 100 30 100 Table 5. Char production at the cyclone outlet (S6) Compound kmol/h kg/h Char 0.29 3.48 T (°C) 800 Table 5. Char production at the cyclone outlet (S6) Table 5. Char production at the cyclone outlet (S6) a percentage of 21%. C. R. Chimie — 2021, 24, nS1, 39-55 3.1. Energy recovery line Before investigating the CHP energy performance, it is fundamental to conduct a preliminary analysis on the physical properties and of the energy content of the obtained gaseous frac- tion at the outlet of the phase separator (S12). The obtained results are summarized in Table 8. Starting from the molar compound, the partial pressure of each compound and the total density of the permanent gas ρpg (i.e. the sum of the partial densities of each gas component) can be calculated. The same approach is used to derive the heat ca- pacity of the gas mixture before condensation, as ex- pressed in (2.6). Finally, the higher and lower heating values have been calculated by (2.9), (2.10) and (2.12). aware choice with respect to the prime motor of the micro-CHP system. As can be observed, the resulting chemicals from oil (Table 6) and gas (Table 7) are comparable; this, as explained by [16], can be since both gaseous and liquid products directly derive from secondary reac- tions of cellulose and hemicellulose, whose degrada- tion mechanism and chemical composition can be considered similar. The available power of the obtained gas can be obtained by multiplying the mass ﬂow rate and the lower heating value LHV [46]. According to the avail- able initial power, the chosen REC2-40G micro-CHP system works at 95.73% of the load. In accordance At 800 °C, carbon monoxide CO is the predom- inant component reaching a fraction of 26%, fol- lowed by carbon dioxide CO2 and hydrogen H2 with C. R. Chimie — 2021, 24, nS1, 39-55 51 Antonio Agrifoglio et al. Table 6. Oil at the phase separator outlet (S16) Compound kmol/h mol% kg/h wt% CO2 8.26×10−6 0.021464 3.63×10−4 0.011451 HCOOH 2.45×10−4 0.636238 0.011265 0.354976 HAA 0.00324 8.421775 0.194564 6.130782 H2 2.44×10−6 0.00635 4.92×10−6 0.000155 CH4 1.70×10−6 0.004406 2.72×10−5 0.000857 HMFU 2.61×10−3 6.778361 3.29×10−1 10.36242 LVG 6.07×10−3 15.7903 9.85×10−1 31.03614 C2H5OH 3.50×10−5 0.091078 0.001614 0.050863 C2H4 9.12×10−7 0.002372 2.56×10−5 0.000807 Coumaryl 0.003232 8.400642 4.85×10−1 15.29314 FE2MACR 5.61×10−4 1.458262 1.17×10−1 3.680663 Phenol 0.007489 19.46657 0.704802 22.20854 Xylosan 1.03×10−3 2.685771 1.37×10−1 4.301363 CH3OH 2.66×10−5 0.069131 0.000852 0.026852 CH2O 2.10×10−5 0.054619 0.000631 0.019881 C3H6O 1.96×10−5 0.050866 1.14×10−3 0.035813 C2H4O 1.60×10−5 0.041485 0.000703 0.022154 Glyoxal 1.84×10−5 0.047958 0.001071 0.03374 CO 2.21×10−6 0.005735 6.18×10−5 0.001947 Char 0.007543 19.60691 0.090598 2.854756 H2O 0.006294 16.3597 0.113382 3.572702 Total 0.038471 100 3.173564 100 T (°C) 65 Table 6. C. R. Chimie — 2021, 24, nS1, 39-55 C. R. Chimie — 2021, 24, nS1, 39-55 3.1. Energy recovery line Oil at the phase separator outlet (S16) sumed to be 5%. Thermal power coming from cool- ing water and exhaust gases is recovered through the two heat exchangers as depicted in Figure 4. The re- covered thermal power values are with the eﬃciencies speciﬁed in the datasheet of the company [45], the theoretical electrical and thermal power are reported in Table 9. The balances used for power generation are based on the scheme represented in Figure 3 [45]. The oxy- gen necessary to complete the combustion of the pyro-gas is calculated by 2.13. According to the liter- ature, the Excess Air Coeﬃcient λ vary between 1.5– 2.5. In this study, λ = 1.8 is assumed as reference value [47–49]. Thus, the derived values used are listed in Table 10. p mcw ·cp,cw·(To,cw −Ti,cw) = mhc ·cp,hc1 ·(T2,hc −T1,hc) = 60.68 (kWth) (3.6) (3.6) mcw ·cp,cw·(To,cw −Ti,cw) = mhc ·cp,hc1 ·(T2,hc −T1,hc) = 60.68 (kWth) (3.7) (3.7) Assuming ηel = 91.2% as energy eﬃciency for the alternator, the resulting electrical power will be Pel = 40.80 (kWel). Mechanical power is obtained by the diﬀerence between the energy content of the pyro-gas, pro- duced thermal power and power losses occurred dur- ing the cycle [25]. The power losses due to mechan- ical friction, heat losses and oil lubrication are as- The Sankey diagram of Figure 8 shows the normal- ized input and output energy ﬂuxes resulting from C. R. Chimie — 2021, 24, nS1, 39-55 52 Antonio Agrifoglio et al. Table 7. Gas at the phase separator outlet (S12) Compound kmol/h mol% kg/h wt% CO2 9.74×10−2 3.505002 4.285882 20.59914194 HCOOH 0.00514 0.185 0.236579 1.137062126 HAA 0.000949 0.034158 0.056993 0.273925665 H2 2.17×100 78.07438 4.372962 21.01767266 CH4 6.85×10−2 2.465883 1.099142 5.282782416 HMFU 5.64×10−9 2.03×10−7 7.12×10−7 3.42072×10−6 C2H5OH 0.001844 0.066353 0.084932 0.408206834 C2H4 9.74×10−3 0.350632 0.273303 1.313570296 Coumaryl 5.32×10−5 0.001915 0.00799 0.038401491 FE2MACR 1.30×10−8 4.67×10−7 2.70×10−6 1.29981×10−5 Phenol 0.00049 0.017644 4.61×10−2 0.221752071 CH3OH 0.002585 0.093037 0.082829 0.398096828 CH2O 0.026022 0.936555 0.781335 3.755314949 C3H6O 4.56×10−3 0.164009 0.264666 1.27205847 C2H4O 0.006173 0.222157 2.72×10−1 1.306919849 Glyoxal 0.002853 0.102666 1.66×10−1 0.795683709 CO 1.88×10−1 6.762725 5.263124 25.29603902 H2O 0.194988 7.017886 3.512771 16.88335526 Total 2.778449 100 20.80612 100 T (°C) 65 Table 7. Gas at the phase separator outlet (S12) Table 7. Gas at the phase separator outlet (S12) Table 8. Physical properties and energy content of the produced gas Table 8. Physical properties and energy content of the produced gas Compound kmol/h kg/h Volume (m3) ρpg (kg/m3) cp,pg (kJ/kg·K) HHV (MJ/kg) LHV (MJ/kg) S12–Gas 2.78 20.81 77.10 0.27 4.06 29.04 23.92 Table 9. Theoretical electrical and thermal power Available initial power Eﬃciency Final power Pg = 138.27 (kW) ηel = 29.5% Pel = 40.79 (kWel) ηth = 62.5% Pth = 86.42 (kWth) the combustion of the syngas in the micro-CHP sys- tem. As can be observed from the diagram, a signiﬁ- cant percentage of thermal recovery can be attained with respect to the initial percentage of biogas and Figure 7. Products distribution at the end of the cycle. being, moreover, the electrical power estimated to be around the 29.5%. /m3) cp,pg (kJ/kg·K) HHV (MJ/kg) LHV (MJ/kg) 7 4.06 29.04 23.92 Figure 7. Products distribution at the end of the cycle. Figure 7. Products distribution at the end of the cycle. the combustion of the syngas in the micro-CHP sys- tem. As can be observed from the diagram, a signiﬁ- cant percentage of thermal recovery can be attained with respect to the initial percentage of biogas and being, moreover, the electrical power estimated to be around the 29.5%. C. R. Chimie — 2021, 24, nS1, 39-55 53 Antonio Agrifoglio et al. Figure 8. Sankey diagram for thermal energy ﬂuxes range of the considered operative parameters, per- manent gas grows at increasing temperature; instead, char and pyro-oil fractions decrease rapidly. Beyond the analysis of the macro-components of pyrolysis, the model is also able to oﬀer a high granularity of the chemical species involved during the ﬁrst and second reactions. This feature can be considered an added value for the proposed modelling methodol- ogy, also in view of the fact that a higher awareness of the composition of chemical species enhances the down-stream decisional process for any energy ap- plication purposes. The proposed case study highlights that the micro-cogeneration unit eﬀectively operates near the full-load, reaching interesting and promising results in terms of both thermal and electrical eﬃ- ciency. In particular, about 62.7% of thermal recovery can be attained with respect to the initial percentage of biogas and being, moreover, the power output esti- mated to be around 30%, which are very promising results. Figure 8. Sankey diagram for thermal energy ﬂuxes. Figure 8. Sankey diagram for thermal energy ﬂuxes. Table 7. Gas at the phase separator outlet (S12) In addition, the chosen system is also able to work in a wide range of load values (until 50%), allowing the implementation of the process even with lower input power. On the other hand, working at lower temperatures reduces the overall performances. Table 10. Air necessary for a complete combustion From the conducted analysis, the choice of the right micro-CHP system depends on the operative parameter of the plant: if it works every time at the same predetermined and standard temperature, it is possible to choose the system concerning that pa- rameter. Otherwise, it is preferable to choose a sys- tem able to work in a wide range of input values, even with reduced eﬃciencies. 4. Conclusions This research proposes a reliable and accurate model aiming at simulating the pyrolysis of biomass. The developed methodology permits to gain awareness on: Further research will include the utilization of ex- perimental data to improve the model and to high- light the dependence from other working parame- ters, such as the heating rate, residence time in the reactor and particle grind size, as well as a sensitiv- ity analysis to deepen how biomass quality and types could aﬀect the results. • The creation of suitable biomass to feed the process; • The prediction of the production yield at the end of the cycle; • The composition of primary products; • The modelling of a micro-CHP system for the valorisation of the produced biogas. C. R. Chimie — 2021, 24, nS1, 39-55 References [14] P. A. Brownsort, “Biomass pyrolysis processes: review of scope, control and variability”, 2009, UKBRC Working paper 5, https://www.biochar.ac.uk/abstract.php?id=16. [37] D. Beckman, D. Elliott, B. Gevert, C. Hörnell, B. Kjellström, A. Östman, Y. Solantausta, V. 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https://openalex.org/W4386621086	https://revistaabalf.com.br/index.html/index.php/rabalf/article/download/745/520	Portuguese	null	FUNDAMENTOS TEÓRICOS SOBRE ALFABETIZAÇÃO E LETRAMENTO NOS CURSOS DE PEDAGOGIA DAS UNIVERSIDADES FEDERAIS DE MINAS GERAIS	Revista Brasileira de Alfabetização	2,023	cc-by	9,029	RESUMO Este estudo tem por centralidade a compreensão de como se organizam as propostas de formação docente dos futuros profissionais que atuarão na alfabetização, na vertente do planejamento curricular dos cursos de graduação em Pedagogia. Diante disso, objetiva-se traçar um panorama das propostas curriculares dos cursos de Pedagogia das Universidades Federais de Minas Gerais acerca das concepções de alfabetização e de letramento, com intuito de dar visibilidade aos aspectos teóricos presentes nos componentes curriculares referentes à formação inicial de professores para atuação na alfabetização. Para tanto, realizou-se uma pesquisa de cunho documental, a partir da análise dos projetos pedagógicos dos cursos (PPC) de Pedagogia das onze Universidades Federais de Minas Gerais, com foco nos ementários e na matriz curricular dos cursos de graduação em Pedagogia, buscando depreender a inserção de referencial teórico alicerçado nas concepções de Magda Soares sobre alfabetização e letramento. As discussões teóricas balizam-se no princípio da formação inicial de Imbernón (2011; 2017), Nóvoa (2019) e Tardif (2014), na concepção de alfabetização e letramento de Soares (2002; 2003; 2004; 2017; 2018). Conclui-se que os projetos pedagógicos de formação de alfabetizadores presentes nos cursos de Pedagogia investigados estão alicerçados nas concepções de alfabetização e de letramento presentes nas obras de Magda Soares, tanto em seus aspectos conceituais quanto práticos. Palavras-chave: Pedagogia. Formação inicial. Currículo. Concepções de Alfabetização e de Letramento. Magda Soares. THEORETICAL FOUNDATIONS ON LITERACY AND LITERACY IN THE PEDAGOGY COURSES OF THE FEDERAL UNIVERSITIES OF MINAS GERAIS Ilsa do Carmo Vieira Goulart Universidade Federal de Lavras ilsa.goulart@ufla.br Giovanna Rodrigues Cabral Universidade Federal de Lavras giovanna.cabral@ufla.br Giovanna Rodrigues Cabral Universidade Federal de Lavras giovanna.cabral@ufla.br FUNDAMENTOS TEÓRICOS SOBRE ALFABETIZAÇÃO E LETRAMENTO NOS CURSOS DE PEDAGOGIA DAS UNIVERSIDADES FEDERAIS DE MINAS GERAIS THEORETICAL FOUNDATIONS ON LITERACY AND LITERACY IN THE PEDAGOGY C THE FEDERAL UNIVERSITIES OF MINAS GERAIS Introdução [...] alfabetizadores(as)dependem do conhecimento dos caminhos da criança – dos processos cognitivos e linguísticos de desenvolvimento e aprendizagem da língua escrita – para orientar seus passos e os passos das crianças [...] Magda Soares (2017, p. 352) Em meio às inquietações que eclodem da ação e atuação docente, especificamente na alfabeti zação, destacam-se as preocupações referentes aos conhecimentos que o professor precisa adquirir sobre a aquisição da escrita, necessários para se garantir uma prática educativa exitosa. Com isso, emergem discussões nas dimensões teórico e metodológica que subsidiam e fundamentam o per curso formativo inicial de professores, capazes de orientar os passos do docente nos “caminhos da criança” para a leitura e da escrita, conforme a epígrafe.i As discussões a respeito da formação docente ramificam-se em duas vertentes: a inicial e a con tinuada, embora ambas se interrelacionam – ou deveriam – percebe-se que abarcam perspectivas teórico e metodológicas diferenciadas, sob legislações específicas e ações formativas que consti tuem uma “forma universitária” (TARDIF; LESSARD, 2014) de gestão, de matrizes curriculares, de programas (ensino, extensão, pesquisa), o que impacta diretamente no perfil de profissional docente. Perpassando o campo intenso e necessário de discussões sobre a formação do professor alfa betizador1, este texto optou pela temática da formação inicial docente, diante da qual buscou-se um olhar mais direcionado aos componentes curriculares dos cursos de graduação em Pedagogia para o processo formativo do professor alfabetizador. Nessa direção, frente à formação inicial para a docência, problematiza-se: como os cursos de graduação organizam sua matriz curricular, de modo a contemplar uma formação que atenda às es pecificidades direcionadas ao processo de alfabetização? Qual fundamentação teórica está prioriza da nos componentes curriculares da área de Alfabetização dos cursos de licenciatura em Pedagogia de universidade federais em Minas Gerais? Diante disso, este texto objetiva traçar um panorama das propostas curriculares dos cursos de Pedagogia das Universidades Federais de Minas Gerais acerca das concepções de alfabetização e letramento, com intuito de dar visibilidade aos aspectos teóricos priorizados no processo de formação de professores alfabetizadores. Para tanto, realizou-se uma pesquisa de cunho documental, a partir da análise dos PPP dos cursos de Pedagogia das onze Universidades Federais de Minas Gerais, com foco nos ementários e nas matrizes curriculares dos cursos de graduação em Pedagogia. ABSTRACT This study has as its centrality the understanding of how the proposals for teacher training of future professionals who will work in literacy are organized, in the aspect of curricular planning of undergraduate courses in Pedagogy. In view of this, the objective is to draw an overview of the curricular proposals of the Pedagogy courses of the Federal Universities of Minas Gerais about the conceptions of literacy and literacy present in the curricular components related to the process of initial training of teachers to act in literacy. To this end, a documentary research was carried out, based on the analysis of the pedagogical projects of the courses (PPC) of Pedagogy of the eleven Federal Universities of Minas Gerais, focusing on the syllabus and curricular matrix of undergraduate courses in Pedagogy, seeking to infer the insertion of a theoretical framework based on Magda Soares’ conceptions of literacy and literacy. The theoretical discussions are based on the principle of the initial formation of Imbernón (2011; 2017), Nóvoa (2019), Tardif (2014), on the conception of literacy and literacy of Soares (2002; 2003; 2004; 2017; 2018). It is concluded that the pedagogical projects of literacy training present in the Pedagogy courses investigated are based on the conceptions of literacy and literacy present in the works of Magda Soares, both in their conceptual and practical aspects. words: Pedagogy. Initial Formation. Curriculum. Conceptions of Literacy and Literacy. Magda So Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 1 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Introdução As discussões teóricas balizam-se no princípio da formação inicial, a partir dos estudos de Imbernón (2011), Nóvoa (1992; 2009; 2017), Pimenta (1999), Gatti (2010; 2014), bem como nas discussões de alfabetização e letramento de Soares (1998; 2003; 2004; 2017; 2020) em interlocução com autores que discutem a temática. Toma-se como um ponto de partida a Resolução CNE/CP nº 2, de 20 de dezembro de 2019, que define as Diretrizes Curriculares Nacionais para a Formação Inicial de Professores para a Educação Básica e institui a Base Nacional Comum para a Formação Inicial de Professores da Educação Básica (BNC-Formação). Com a promulgação da Resolução CNE/CP nº 2/2019, retomam-se as discussões no âmbito da formação inicial de professores (GATTI, 2010; 2014) referentes à docência superior dos 1 A formação continuada do professor alfabetizador, nos últimos anos, assume vertentes distintas de reflexão, no âmbito dos programas federais como o PNAIC, um campo de discussões das políticas públicas (SILVA, SIGNORELLI, 202; MARTINS FILHO; BARROS-MENDES; OLIVEIRA, 2022) como das percepções de docentes sobre saberes (PINTO; GOULART; CABRAL, 2021; GOULART, RAMOS, CABRAL, 2023; AZARIAS, GOULART, CABRAL, 2023), ou mesmo das práticas pedagógicas (CABRAL, GOULART, AQUINO, 2022; CABRAL, GOULART, NAVES, 2022). Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 2 cursos de licenciatura (CUNHA; ZANCHET; RIBEIRO, 2013), e reflexões sobre quais saberes ou conhe cimentos pedagógicos prioritários (PIMENTA, 1999) podem garantir uma melhor qualificação dos pro fissionais para atuar na educação básica. Embora as pesquisas referentes à formação docente tenham obtido uma ascensão considerável nas últimas décadas, como apontam os estudos de André (2009; 2010), percebe-se a necessidade de refletir a respeito das especificidades de conhecimentos de área a fim de proporcionar ações formativas condizentes com as reais necessidades do contexto educativo.i O documento da BNC-Formação (BRASIL, 2019), a fim de compor tais diretrizes curriculares para se constituir uma base nacional de formação de licenciandos, toma como referência a implantação da Base Nacional Comum Curricular da Educação Básica (BNCC), instituída pelas Resoluções CNE/CP nº 2/2017 e CNE/CP nº 4/2018. Um documento que articula a formação curricular a partir das compe tências dos estudantes. Seguindo essa vertente, o Art. 2º, da Resolução CNE/CP nº 2/2019 estabelece dez competên cias gerais e específicas, esta última abarca três dimensões: como conhecimento profissional, prática profissional e engajamento profissional, as quais subdividem-se em habilidades que devem ser asse guradas no processo formativo de professores. Introdução Estudos recentes demonstram que a implementação da BNC-Formação pode incidir em mudan ças significativas para o curso de Pedagogia, de inserção de aspectos voltados às formações espe cíficas e maior foco às atividades práticas emergentes à atuação docente, o que pode “representar um divisor de águas para o curso, posto que esse ainda parece manter estruturas baseadas em regu lamentações e paradigmas já superados, o que reforça as limitações historicamente diagnosticadas” (SILVA; GUILHERME; BRITO, 2023, p.13). Para organização do texto, apresenta-se uma seção a respeito da formação inicial docente di recionada à reflexão sobre o professor alfabetizador, outra que abarca as fundamentações teóricas sobre alfabetização e letramento, para em seguida trazer os aspectos metodológicos e os resultados obtidos com a pesquisa. Nesse sentido, ressalta-se o panorama dos Cursos de Pedagogia, regidos por Diretrizes específi cas (BRASIL, 2006) e, agora sob risco de alterações profundas em sua conformação, a partir da publi cação das Diretrizes voltadas para a formação de professores (2019), alicerçadas nas competências e habilidades de ensino previstas na BNCC (BRASIL, 2017). Essa política sinaliza para a formação reduzida à prática na escola, como se o trabalho docente se limitasse à transmissão de informações, na maioria das vezes isenta de um compromisso social. Nesta atual perspectiva não há compromisso com a pesquisa e a produção de conhecimentos, o que nos traz desafios quando pensamos na di mensão reflexiva necessária à função docente.li Assim, a formação do professor, em uma dimensão reflexiva, é fundamental para a qualificação do trabalho pedagógico. Defende-se que a formação inicial associada à continuada, e ao compromis so político e ético do profissional com sua escola como condição para a construção de uma identida de docente sólida e para o desenvolvimento profissional do professor. A formação inicial significa a aquisição, por parte dos professores, dos conhecimen tos necessários para o exercício da atividade pedagógica, construída pela agência formadora, e a formação continuada significa o processo dinâmico através do qual, no exercício de sua prática profissional, os professores vão adequando sua formação às exigências das condições concretas em que estão situados (CABRAL, 2010, p.14). Assim, pensar a formação docente voltada para a etapa da alfabetização significa garantir um repertório de saberes múltiplos para subsidiar essa atuação, sobretudo pelo cenário de não apren dizagem indicado pelas avaliações externas, associada aos efeitos da suspensão das aulas pre senciais nas escolas devido à pandemia. Os dados do Sistema de Avaliação da Educação Básica (SAEB, 2021) mostram que houve queda de desempenho dos estudantes, que demarca o impacto da Covid-19 nas escolas do país. A maior queda foi, exatamente na etapa da alfabetização – leitura e escrita no 2º ano do ensino fundamental, onde a média de 750 em 2019 caiu para 726, cerca de meio desvio padrão, o que caracteriza uma queda na proficiência muito grande. Para reverter esse quadro não basta apenas pensar na formação de professores, é preciso em uma robusta política pública voltada para a alfabetização, com indicação de quais são as concep ções nas quais ela se baseia, nos resultados que se deseja atingir, no percurso a ser delineado para implementá-la e no compromisso dos entes públicos para sua execução. Entre as discussões sobre a formação inicial docente: uma reflexão sobre o professor alfabetizador Sabe-se que a formação docente, essencial para a qualidade do ensino, considera um conjunto de saberes alicerçados em aspectos teóricos e prática, que se apoiam nas histórias de vida, no de senvolvimento profissional e institucional como constituintes da identidade do professor. Essa formação inicial de professores no Brasil respondeu ao modelo de expansão do ensino superior, iniciado em 1990, no âmbito das reformas do Estado, com a diversificação e a flexibilização da oferta de cursos, como Normal superior, Pedagogia, Licenciaturas, cursos de complementação pedagógica e cursos à distância - de modo a atender a demanda e a necessidade de qualificação daqueles profissionais que já atuavam nas escolas. A oferta de formação inicial, concentrada em instituições privadas que, muitas vezes, desconsi deram os preceitos da qualidade do ensino, alicerçada conjuntamente com a pesquisa e a extensão e o aumento do número de cursos a distância voltados para a docência, refletem condições desiguais aos processos de formação de professores. Somado a isso, a manutenção da oferta de cursos téc nicos em nível médio, específicos para a docência na educação infantil e nos anos iniciais do ensino fundamental, consolidam o cenário de formação para atuação na etapa da alfabetização como políti ca pública permanente, não transitória. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 3 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 da natureza simbólica da escrita; distância entre a variedade escrita e a variedade oral das crianças; diferentes funções atribuídas à leitura e à escrita pelos diversos setores sociais; conflito entre o con texto cultural familiar e a cultura da escola; dificuldades para definir com clareza o que uma criança deve adquirir e dificuldades para trabalhar na sala de aula, com a diversidade cultural e os ritmos de aprendizagem. Se os fatores causais são muitos, no entanto, uma coisa é certa a não aprendizagem da leitura e da escrita afeta sobretudo as crianças das classes populares. Os problemas de infraestrutura de ensino adequada, de falta de professores preparados, de políticas educacionais eficazes, de recursos, no geral, também são apontados responsáveis pela situação de fracasso na alfabetização. No entanto, pode-se depreender que a baixa proficiência de crianças e jovens em leitura pode ser associada, em algumas situações, às tentativas malsucedidas de implementar uma concepção mais atualizada de ensino, baseada na associação dos conceitos de apropriação do sistema de escrita e de letramento. Na década de 80 vigoravam no Brasil as concepções tradicionais de alfabetização, centradas na ideia de que a escrita se limitava a um código de representação da linguagem oral. Nesta concep ção as cartilhas figuravam como norteadoras do processo de ensino, já que o foco dos educadores se voltava para a forma, para os signos e sinais. Não havia uma preocupação com o entendimento do significado e com contextualização da leitura e da escrita, por isso, as crianças eram expostas a textos artificias e desprovidos de sentido para um aprendizado mecânico da língua. Passou-se a compreensão de que as concepções e os métodos tradicionais de alfabetização eram falhos – pois visavam, essencialmente, à codificação e decodificação – e, para responder às demandas sociais, que exigiam habilidades mais amplas, era preciso pensar uma nova relação com a leitura e a escrita, dentro da perspectiva do letramento, cuja concepção enfatiza o uso social e a dimensão simbólica dos textos, sendo os seus significados – determinados histórica e culturalmente – construídos socialmente. Essa ampliação e associação da alfabetização a partir do letramento reforça que o problema não é apenas ensinar a criança os códigos, mas também as práticas e usos sociais da escrita. A fundamentação teórica sobre alfabetização e letramento na formação inicial Além da alfabetização, foco do trabalho na pré-escola e nos anos iniciais do ensino fundamen tal, o letramento tornou-se uma necessidade de ser incorporada nas práticas de leitura e escrita das escolas. Isso se deu, principalmente, em decorrência da produção de pesquisadores como Soares (1998) e Kleiman (1995) para quem não basta alfabetizar, é preciso letrar a população, para que os indivíduos possam usar a leitura e a escrita como instrumento de informação e formação, tanto no meio profissional, quanto na esfera familiar e social. Soares (1998, p. 18) define letramento como “[...] o resultado da ação de ensinar ou de aprender a ler e escrever: o estado ou a condição que adquire um grupo social ou um indivíduo como consequência de ter-se apropriado da escrita”. i Soares (1998; 2004) aponta que muitas dificuldades na etapa de alfabetização são marcadas por diferentes entendimentos ao longo da história educacional brasileira: falta de prontidão da criança; ca rência e diferença cultural; reprodução das desigualdades sociais; diferentes níveis de compreensão Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 4 No Brasil essa perspectiva refletiu no trabalho dos professores que, ansiosos por abandonar o modelo das cartilhas – mas sem compreender as reais dimensões do letramento –, acabaram por sobrepor ao conceito de alfabetização, como se eles fossem antagônicos. Essa “curvatura da vara” levou à ideia equivocada de que a alfabetização não necessita de um trabalho pedagógico sistemáti co: bastaria expor a criança a uma grande quantidade e variedade de materiais escritos, fazê-la ouvir histórias, interagir com textos para que ela aprendesse a ler. Na verdade, trata-se de um equívoco, pois é sabido que a alfabetização não acontece de forma espontânea na criança e que é preciso um trabalho sistematizado do professor para que ela conheça e se aproprie do sistema de escrita alfa bético. Dessa forma, os cursos de formação inicial voltados para a formação de alfabetizadores preci sam agregar em suas propostas pedagógicas componentes curriculares que resgatem a sistemati zação dos conteúdos da linguagem. Os cursos de Pedagogia atentam-se para a inserção e um com ponente curricular que discute sobre o processo de aquisição do sistema convencional alfabético e ortográfico, necessário para o uso funcional da escrita, de modo a associar às práticas e usos sociais destes conhecimentos para a inserção social qualificada das crianças e jovens no mundo da leitura e escrita. Tal inserção do componente curricular se mostra um elemento determinante e ao mesmo tempo desafiador na formação inicial de professores alfabetizadores. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 5 Aspectos metodológicos Para o alcance do objetivo - traçar um panorama das propostas curriculares dos cursos de Pedagogia das Universidades Federais de Minas Gerais acerca das concepções de alfabetização e de letramento presentes nos componentes curriculares afetos ao processo de formação inicial de professores para atuação na alfabetização - realizou-se uma pesquisa de cunho documental, a partir da análise dos projetos pedagógicos dos cursos (PPC) de Pedagogia das Universidades Federais de Minas Gerais, com foco nos ementários e nas matrizes curriculares dos cursos de graduação em Pedagogia, buscando depreender se e como acontece a inserção de referencial teórico alicerçado nas concepções de Magda Soares sobre alfabetização e letramento. Quando se observa a distribuição de universidades federais pelo Brasil, nota-se que Minas Ge rais é um Estado brasileiro atípico. Isso porque é o Estado da Federação com o maior número de universidades públicas federais do país, representando 20% do quantitativo do Brasil. No total, o país apresenta 55 universidades federais e, em Minas Gerais encontram-se 11 dessas, valor esse superior ao número de universidades federais das regiões Norte (com oito universidades federais), Sul (com nove universidades federais) e Centro-Oeste (com cinco universidades federais). Quadro 1 - Universidades Federais de Minas Gerais, sigla e localização do campus sede Quadro 1 - Universidades Federais de Minas Gerais, sigla e localização do campus sede Universidades Sigla Localização - região Universidade Federal de Juiz de Fora UFJF Zona da Mata Universidade Federal de Lavras UFLA Campo das vertentes Universidade Federal de Minas Gerais UFMG Região metropolitana Universidade Federal de Ouro Preto UFOP Central Universidade Federal de São João del-Rei UFSJ Campo das vertentes Universidade Federal do Triângulo Mineiro UFTM Triângulo mineiro Universidade Federal de Uberlândia UFU Triângulo mineiro Universidade Federal de Viçosa UFV Zona da Mata Universidade Federal dos Vales do Jequitinhonha e do Mucuri UFVJM Jequitinhonha Universidade Federal de Alfenas UNIFAL Sul de Minas Universidade Federal de Itajubá UNIFEI Sul de Minas A figura 1 retrata o mapa de Minas Gerais dividido pelas regiões que compõem a organização administrativa do Estado. A partir da localização das universidades federais de Minas Gerais perce be-se que há uma boa distribuição geográfica no estado. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 6 Figura 1 - Mapa das regiões de Minas Gerais Figura 1 - Mapa das regiões de Minas Gerais Fonte: Semesp – 11º Mapa do Ensino Superior (2023) Fonte: Semesp – 11º Mapa do Ensino Superior (2023) Fonte: Semesp – 11º Mapa do Ensino Superior (2023) Fonte: Semesp – 11º Mapa do Ensino Superior (2023) Além do campus sede, as universidades federais de Minas Gerais atuam em outras cidades do Estado, ampliando a interiorização e oportunizando o acesso ao ensino superior a população mineira, por meio da oferta de cursos presenciais em diferentes áreas do conhecimento e da presença de polos da Universidade Aberta do Brasil (UAB) para oferta de cursos na modalidade a distância. O quadro 2 apresenta as cidades sede e os campi de cada instituição de ensino superior (IES) de Minas Gerais Quadro 2 – Cidades sede e campi das Universidades federais de Minas Gerais Quadro 2 – Cidades sede e campi das Universidades federais de Minas Gerais UFJF Juiz de Fora e Governador Valadares UFLA Lavras e São Sebastião do Paraíso UFMG Belo Horizonte e Montes Claros UFOP Ouro Preto, Mariana e João Monlevade UFSJ São João del-Rei, Ouro Branco, Divinópolis e Sete Lagoas UFTM Uberaba e Iturama UFU Uberlândia, Ituiutaba, Monte Carmelo e Patos de Minas UFV Viçosa, Florestal e Rio Paranaíba UFVJM Diamantina, Janaúba, Teófilo Otoni e Unaí UNIFAL Alfenas, Poços de Caldas e Varginha UNIFEI Itajubá e Itabira Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 7 Das 11 universidades federais mineiras, apenas uma não oferta o curso de Pedagogia presencial ou a distância, trata-se da UNIFEI. E, das 10 universidades que possuem curso de Pedagogia, todas ofertam o curso presencialmente e 6 ofertam além do presencial a modalidade a distância. Pelo levan tamento realizado, percebe-se que a maioria dos cursos organizam os componentes curriculares de Alfabetização até o 5º período, apenas a Pedagogia presencial da UFLA oferta os componentes no 7º período e a Pedagogia a distância da UNIFAL no 6º período. Quadro 3 - Distribuição dos componentes da área de Alfabetização nos cursos de Pedagogia das IES Universidades Cursos Período Componente Curricular CH UFMG Pedagogia presencial Diurno e Noturno 3º Alfabetização e Letramento I 60 4º Alfabetização e Letramento II 60 UFLA Pedagogia presencial Noturno 7.º Alfabetização e Letramento 85 7.º Práticas de Alfabetização e Letramento 34 Pedagogia a distância 3º Alfabetização e Letramento em Língua Portuguesa: aspectos teóricos e práticos 90 UFOP Pedagogia presencial Noturno 4º Alfabetização e letramento I 60 5º Alfabetização e letramento II 60 Pedagogia a distância 3º Ensino e aprendizagem de linguagem I 60 4º Ensino e aprendizagem de linguagem II 60 UFJF Pedagogia presencial Diurno e Noturno 3º Fundamentos Teóricos Metodológicos em Alfabetização I 60 Eletiva Fundamentos Teóricos Metodológicos em Alfabetização I 60 Pedagogia a distância 2º Alfabetização e letramento 60 UFSJ Pedagogia Presencial Noturno 5º Fundamentos e didática da alfabetização 72 UNIFAL Pedagogia Presencial 3º Alfabetização e Letramento I 60 4º Alfabetização e Letramento II 60 Pedagogia a distância 5º Fundamentos e Metodologia de Alfabetização I 60 6º Fundamentos e Metodologia de Alfabetização II 60 UFV Pedagogia Presencial 2º Alfabetização e Letramento 60 UFVJM Pedagogia Presencial 3º Alfabetização e Letramento 75 Pedagogia a distância 5º Alfabetização e Letramento I 60 UFU Pedagogia Presencial 3º Princípios e Métodos de Alfabetização 90 Pedagogia a distância 3º Processo de Alfabetização I 60 4º Processo de Alfabetização II 60 UFTM Pedagogia Presencial 2º Escrita, alfabetização e letramento 75 Quadro 3 - Distribuição dos componentes da área de Alfabetização nos cursos de Pedagogia ribuição dos componentes da área de Alfabetização nos cursos de Pedagogia das IES 8 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 8 Pelo quadro 3 depreende-se que o maior somatório de carga horária dos componentes das áreas de alfabetização nos cursos de Pedagogia chega a 120 horas e a menor 60 horas, o que equivale a, aproximadamente, 3,75% e 1,88%, respectivamente, da carga horária mínima definida pelas DCN’s (2006) específicas do curso de Pedagogia. Isso aponta para o pouco contato dos estudantes com as discussões teóricas e práticas sobre a docência na alfabetização. Além disso, foram encontrados 24 componentes curriculares ofertados nos cursos de Pedago gia das instituições pesquisadas, sendo que 14 deles fazem menção expressa ao nome alfabetiza ção e ao letramento. Três instituições ofertam apenas 1 disciplina sobre alfabetização nos cursos de Pedagogia. No quadro 6 destaca-se os componentes curriculares com as descrições de suas respectivas ementas. Quadro 4 - Ementas dos componentes da área de Alfabetização nos cursos de Pedagogia Universidades Cursos Componente Curricular Ementa do componente UFMG Pedagogia Alfabetização e Letramento I Conceituação de alfabetização e letramento. Fa cetas social, histórica, antropológica, linguística, sociolinguística e psicolinguística dos processos de alfabetização. Diferentes matrizes teóricas sobre aprendizado da leitura e da escrita e suas implicações pedagógicas nos diferentes níveis de ensino. Alfabetização e Letramento II Condições escolares de ensino-aprendizagem da escrita na Educação Infantil, no Ensino Funda mental, e na Educação de Jovens e Adultos. Ca pacidades linguísticas envolvidas no processo de aquisição da escrita. Planejamento e organização dos processos de alfabetização e letramento. UFLA Pedagogia Alfabetização e Letramento Reflexão sobre as diferentes concepções de al fabetização e letramento. Considerações sobre o processo de aquisição da linguagem escrita pela criança. Estudo dos processos cognitivos, sociais e interativos que envolvem o desenvolvimento da competência leitora e escritora nas crianças. Práticas de Alfabetização e Letramento Reflexão teórico-prática sobre o processo de en sino e de aprendizagem do sistema de escrita alfabética. Estudo sobre as intervenções peda gógicas e o processo de aquisição da linguagem escrita pela criança. Discute alternativas metodo lógicas para a prática pedagógica nos processos de alfabetização e de letramento na escola. Pedagogia a distância Alfabetização e Letramento em Língua Portuguesa: aspectos teóricos e práticos Reflexão sobre as diferentes concepções de al fabetização e letramento. Considerações sobre o processo de aquisição da linguagem escrita pela criança. Estudo dos processos cognitivos, sociais e interativos que envolvem o desenvolvimento da competência leitora e escritora nas crianças. UFOP Pedagogia Alfabetização e letramento I Linguagem e alfabetização: diferentes concepções teóricas (oralidade e escrita); alfabetização e letra mento: discussão conceitual; visão histórica e análi se crítica dos métodos de alfabetização tradicionais (analíticos e sintéticos); Psicogênese da Língua escrita; fundamentos linguísticos da alfabetização, diagnóstico inicial da alfabetização; análise de livros e materiais didáticos para alfabetização Alfabetização e letramento II Eixos do processo de alfabetização; capacidades linguísticas a serem desenvolvidas pelos alfabeti zandos na apropriação do sistema de escrita alfa bética; planejamento do processo de alfabetização; elaboração de rotinas e atividades para a apropria ção do sistema de escrita e para o desenvolvimento das habilidades de leitura e oralidade. Acompanha mento e avaliação da aprendizagem em alfabetiza ção; alfabetização. Pedagogia a distância Ensino e aprendizagem de linguagem I Reflexão conceitual sobre letramento e alfabetização e os desafios que o trabalho na perspectiva do letra mento impõe aos professores do ensino fundamental especificamente. Abordagem da relação letramento, alfabetização e diversidade textual bem como o que caracteriza o indivíduo alfabetizado e letrado. As difi culdades de aprendizagem: contribuições do campo de estudos da psicologia e principalmente do campo de estudos da educação e linguagem. Ensino e aprendizagem de linguagem II Aquisição e desenvolvimento da linguagem. Articu lação entre língua falada e língua escrita. Produção oral e escrita. Geração de textos criativos. Leitura e produção de texto, metodologia e estratégias es pecíficas. Os Parâmetros curriculares Nacionais e o ensino de Linguagem. UFJF Pedagogia Fundamentos Teóricos Metodológicos em Alfabetização I Conceitos básicos na área da aquisição e aprendiza gem da língua escrita; Fundamentos do processo de ensino/aprendizagem da língua escrita; Alfabetização e letramento na história da escrita e no quadro dos diferentes sistemas de escrita; Consequências, para grupos sociais e para o indivíduo, do acesso à escri ta; Processos de aquisição do sistema de escrita e o processo de aprendizagem do sistema ortográfico; A questão dos métodos de alfabetização e diretrizes metodológicas para o processo de alfabetização; Ma teriais didáticos destinados à alfabetização. Fundamentos Teóricos Metodológicos em Alfabetização I Leitura e análise crítica de documentos oficiais con cernentes à alfabetização e à formação do(a) profes sor(a) alfabetizador(a). Planejamento do processo de alfabetização escolar. Elaboração de material al ternativo para o ensino da leitura e da escrita inicial. O texto literário na alfabetização. Pedagogia a distância Alfabetização e letramento Diferentes perspectivas de alfabetização e letramen to. 9 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 9 A relevância da perspectiva social, cognitiva e dis cursiva de linguagem. Aquisição e desenvolvimento da linguagem. Articu lação entre língua falada e língua escrita. Produção oral e escrita. Geração de textos criativos. Leitura e produção de texto, metodologia e estratégias es pecíficas. Os Parâmetros curriculares Nacionais e o ensino de Linguagem. Diferentes perspectivas de alfabetização e letramen to. A relevância da perspectiva social, cognitiva e dis cursiva de linguagem. 10 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 10 UFSJ Pedagogia Fundamentos e didática da alfabetização Conceitos de alfabetização, conceitos linguísticos básicos (principalmente de fonética e fonologia), teorias de aquisição da linguagem oral, questões da diversidade linguística de grupos sociais e questões da análise de erros, visando a formação de uma profissão capaz de desenvolver sua prá tica alfabetizadora fundamentada em referenciais da teoria linguística. UNIFAL Pedagogia Alfabetização e Letramento I A aprendizagem da escrita pela criança: as con tribuições das pesquisas de Emília Ferreiro (Psi cogênese) e Alexander Luria. A aprendizagem da leitura: concepções e fundamentos. O ensino da alfabetização e suas interfaces com o letramen to: contextualização histórica da área de alfabe tização. Percurso das metodologias de alfabeti zação. O uso das cartilhas e dos atuais livros de alfabetização. O conceito de letramento. O texto como unidade de ensino. Alfabetização e Letramento II Abordagem prática da alfabetização e letra mento. Criação de um Ambiente alfabetizador. O uso dos diversos gêneros textuais: a escrita como produção social. Análise e planejamento de situações didáticas e experiência no cotidia no escolar. O uso da biblioteca escolar. Estu dos de Propostas Curriculares e das Avaliações Governamentais para a área de alfabetização e letramento. Análise e produção de material di dático. Pedagogia a distância Fundamentos e Metodologia de Alfabetização I Contextualização histórica da área de alfabetiza ção. Percurso das metodologias de alfabetização. O uso das cartilhas e livros de alfabetização. Aqui sição da linguagem escrita a partir das contribui ções de Emília Ferreiro e L. S. Vigotski. O texto como unidade de ensino. Perspectivas teóricas sobre o conceito de leitura. O conceito de letra mento Fundamentos e Metodologia de Alfabetização II Abordagem prática da alfabetização e letra mento. Ambiente alfabetizador. O uso dos di versos gêneros textuais: a escrita como produ ção social. Análise e planejamento de situações didáticas e experiência no cotidiano escolar. O uso da biblioteca escolar. Análise e produção de material didático. Estudos de Propostas Cur riculares UFV Pedagogia Presencial Alfabetização e Letramento Referencial Curricular Nacional para a Educação Infantil. Alfabetização e Letramento. Métodos de alfabetização. Aquisição da língua escrita. Am biente alfabetizador. Conceitos de alfabetização, conceitos linguísticos básicos (principalmente de fonética e fonologia), teorias de aquisição da linguagem oral, questões da diversidade linguística de grupos sociais e questões da análise de erros, visando a formação de uma profissão capaz de desenvolver sua prá tica alfabetizadora fundamentada em referenciais da teoria linguística. 11 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 11 UFVJM Pedagogia Alfabetização e Letramento Alfabetização e letramento: conceitos e implicações pedagógicas. Aquisição da linguagem oral e escri ta. Alfabetização e letramento enquanto construção cognitiva, histórica, psicolinguística, sociolinguísti ca. Diferentes teorias sobre o processo de apren dizagem da língua escrita. Métodos de alfabetiza ção. Produção textual na infância e na Educação de Jovens e Adultos em diferentes gêneros textuais. Leitura de diferentes gêneros textuais. Prática Pe dagógica no ensino da língua escrita Pedagogia a distância Alfabetização e Letramento I Estudo da alfabetização, letramento e cultura escri ta. O processo de construção/aquisição da leitura e da escrita. Estudo e análise dos métodos de al fabetização. Aspectos linguísticos, sociolinguísticos e psicolinguísticos no processo de alfabetização. Análise e produção de materiais didáticos para a Alfabetização UFU Pedagogia Princípios e Métodos de Alfabetização Funções sociais da lecto-escrita. O processo de construção e aquisição da leitura e da escrita. Estu do e análise dos métodos de alfabetização. Análise e produção de materiais didáticos para a Alfabeti zação. O Pedagogo e o processo de alfabetização. Pedagogia a distância Processo de Alfabetização I História da Alfabetização no Brasil. Métodos de al fabetização. O processo de aquisição da leitura e da escrita segundo a Psicogênese da Língua Es crita. A alfabetização enquanto processo discursi vo. Análise de projetos, currículos e programas de alfabetização. Processo de Alfabetização II Conceitos de alfabetização e letramento. A alfabe tização enquanto ato político. A alfabetização sob a perspectiva da linguística. Análise e construção de materiais didáticos para a alfabetização. UFTM Pedagogia Escrita, alfabetização e letramento Alfabetização, Leitura e Linguagem: Alfabetização. A alfabetização no contexto histórico. Sujeitos da alfabetização. Leitura e Linguagem. As referências básicas e complementares dos componentes curriculares analisados trazem uma variedade de obras, dentre as quais se despontam os estudos de Emília Ferreiro e Ana Tebe rosky sobre a Psicogênese da língua escrita, os estudos do conceito de letramento proposto por Magda Soares e os estudos do sistema de escrita alfabética de Artur Morais. Ao analisar as referências complementares dos 24 componentes curriculares listados, 8 não apresentam obras da Magda Soares na relação de referências básicas. Contabilizando referên cias básica e complementares, apenas 4 componentes não relacionam obras de Magda Soares, como UFLA (Práticas de Alfabetização e Letramento); UFVJM (Alfabetização e Letramento I), UFU (Processo de Alfabetização I) e UFSJ (Fundamentos e didática da alfabetização). Verificou-se que apenas uma instituição não cita obras de Magda Soares nem apresenta disciplina da área de alfabetização - UFSJ - (Fundamentos e didática da alfabetização). A inserção das discussões referentes ao letramento nas ementas dos componentes curri culares dos cursos de Pedagogia demonstra certa preocupação com a formação do professor alfabetizador, na medida em que “[...] não se trata de convocar apenas as questões práticas ou a preparação profissional, no sentido técnico ou aplicado, mas de compreender a complexidade da profissão em todas as suas dimensões (teóricas, experienciais, culturais, políticas, ideológicas, simbólicas, etc.)”, conforme discute Nóvoa (2019, p. 7). Pensar na dimensão formativa para a atuação na alfabetização é pensar na perspectiva de articulação entre os aspectos teóricos e práticos da aquisição a língua escrita. A formação de conhecimentos teóricos e conceituais que fica a cargo da universidade, a qual delibera, seleciona e privilegia temas, “define um campo estimulante” (NÓVOA, 2019, p. 8) prioritário para a formação profissional. Tais conhecimentos precisam dialogar com a realidade escolar e este esforço se percebe em componentes que se subdividem (I e II) para assegurar a proximidade entre teoria e prática pedagógica. Tal dimensão teórica é basilar para se pensar a educação, entretanto sua discussão precisa estar embasada na dimensão prática e evitar “[...] mucha palabrería teórica – en formato artículo con objeto de aumentar el currículo personal, aunque por supuesto siempre hay excepciones – que dificilmente se convirte en una práctica en la formación inicial” (IMBER NÒN, 2017, p. 60). O desenvolvi mento da leitura no Brasil. Interação de leitura no contexto social. O prazer da leitura. Produção Tex tual e Letramento. A evolução da escrita. A prática pedagógica na produção textual. Escrita de textos na educação infantil. A importância da escrita para o homem. Letramento: Alfabetização e letramento: Teóricos e teorias do letramento. Fonte: Dados da pesquisa. Fonte: Dados da pesquisa. Pode-se ressaltar, a partir do quadro, dois aspectos referentes à estruturação da matriz cur ricular: no primeiro se evidencia a distribuição de dois componentes curriculares para abarcar as discussões acerca da alfabetização, que aparecem distribuídos em disciplinas que expressam a continuidade da temática (I e II); no segundo observa-se que as ementas trazem, em sua maioria, a discussão sobre letramento e alfabetização, que ora aparecem na dimensão conceitual ou de concepção teórica, ora estendem-se às práticas do letramento. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 12 Em relação aos aspectos formativos nos componentes curriculares que estão direcionados à formação para a atuação na alfabetização, eles apresentam bases teóricas a partir da seleção de obras que discutem a aquisição da língua escrita em diferentes perspectivas e diferentes auto res são listados. Entretanto, para este estudo, optou-se por listar as obras de Magda Soares que constam nas referências básicas e complementares dos componentes da área de alfabetização, conforme observa-se no Quadro 7. Quadro 7 - Lista das obras de Magda Soares que constam nas referências básicas e complementares dos componentes da área de alfabetização Componentes Referência básica Referência complementar UFMG Alfabetização e Letramento I SOARES, Magda Becker. Letramento e alfabetização: as muitas facetas. Revista Brasileira de Educação, n.24, jan./fev./mar./abr. 2004. SOARES, Magda. Livro: Alfabetização: a questão dos métodos. São Paulo: Cortez, 2016 Alfabetização e Letramento II Não consta SOARES, Magda. Linguagem e escola: uma perspectiva social. São Paulo: Ática, 2002. Quadro 7 - Lista das obras de Magda Soares que constam nas referências básicas e complementares dos componentes da área de alfabetização Componentes Referência básica Referência complementar UFMG Alfabetização e Letramento I SOARES, Magda Becker. Letramento e alfabetização: as muitas facetas. Revista Brasileira de Educação, n.24, jan./fev./mar./abr. 2004. SOARES, Magda. Livro: Alfabetização: a questão dos métodos. São Paulo: Cortez, 2016 Alfabetização e Letramento II Não consta SOARES, Magda. Linguagem e escola: uma perspectiva social. São Paulo: Ática, 2002. Quadro 7 - Lista das obras de Magda Soares que constam nas referências básic e complementares dos componentes da área de alfabetização 13 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 13 UFJF Fundamentos Teóricos Metodológicos em Alfabetização I SOARES, Magda. A reinvenção da alfabetização. Presença Pedagógica, Belo Horizonte, v. 9, n.º 52, p.1-7, 2003. SOARES, Magda. Alfabetização. In: FRADE, Isabel Cristina Alves da Silva et al. Glossário Ceale: termos de alfabetização, leitura e escrita. Belo Horizonte: Ceale/ FaE/ UFMG, 2014. SOARES, Magda. Alfabetização: a questão dos métodos. São Paulo: Contexto, 2016. SOARES, Magda. Alfaletrar: toda criança pode aprender a ler e a escrever. São Paulo: Contexto, 2020. SOARES, Magda. Letramento. In: FRADE, Isabel Cristina Alves da Silva et al. Glossário Ceale: termos de alfabetização, leitura e escrita. Belo Horizonte: Ceale/ FaE/ UFMG, 2014. SOARES, Magda. Alfabetização e letramento. São Paulo: Contexto, 2010. SOARES, Magda. Letramento e Alfabetização: as muitas facetas. Revista Brasileira de Educação, n.º 25, p. 5-17, Jan./Abr. 2004. Fundamentos Teóricos Metodológicos em Alfabetização II SOARES, Magda B. Aprender a escrever, ensinar a escrever. In: TOZZI, Devanil A.& ONESTI, Lizete Freire (Orgs.). Os desafios enfrentados no cotidiano escolar. São Paulo: FDE, 1997. p. 59-75. SOARES, Magda B. A (des)aprendizagem das funções da escrita. Educação em Revista, Belo Horizonte: Faculdade de Educação da UFMG, n. 8, p. 3-11, dez. 1988. SOARES, Magda B. As muitas facetas da alfabetização. Cadernos de Pesquisa, São Paulo, n. 52, p. 19-24, fev. 1985. não indicado Alfabetização e letramento SOARES, Magda. Alfabetização: acesso a um código ou acesso à leitura? ONG Leia Brasil. Rio de Janeiro. SOARES, Magda. A reinvenção da alfabetização. Disponível em: http://www.meb. org.br/biblioteca/artigomagdasoares. SOARES, Magda. Letramento e alfabetização: as muitas facetas. Revista Brasileira de Educação. Jan. /Fev./ Mar./Abr. 2004. n. 25. SOARES, Magda. Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 2003. não indicado UFSJ Fundamentos e didática da alfabetização Não consta Não consta UNIFAL Alfabetização e Letramento I Indicação de referências não localizada Indicação de referências não localizada Alfabetização e Letramento II Indicação de referências não localizada Indicação de referências não localizada Fundamentos e Metodologia de Alfabetização I Não consta SOARES, Magda. Letramento e alfabetização. São Paulo: Contexto: 2008. Fundamentos e Metodologia de Alfabetização II SOARES, Magda. Letramento: um tema em três gêneros. 4. ed. Belo Horizonte: Autêntica, 2010. não indicado UFV Alfabetização e Letramento SOARES, Magda. Alfabetização e Letramento. São Paulo, Contexto, 2003. SOARES, Magda. Letramento: um tema em três gêneros. Belo Horizonte, Autêntica, 2001. Não consta SOARES, Magda. Alfabetização e letramento. São Paulo: Contexto, 2010. SOARES, Magda. Letramento e Alfabetização: as muitas facetas. UFLA Alfabetização e Letramento SOARES, Magda. Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 2001. SOARES, Magda. Alfabetização e Letramento. São Paulo: Contexto, 2003. Práticas de Alfabetização e Letramento Não consta Não consta Alfabetização e Letramento em Língua Portuguesa: aspectos teóricos e práticos SOARES, Magda. Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 2001. SOARES, Magda. Alfabetização e Letramento. São Paulo: Contexto, 2003. UFOP Alfabetização e letramento I SOARES, M. B. Letramento e Alfabetização: as muitas facetas. Rev. Brasileira Educação Rio de Janeiro, n.25, 2004, p.5-14. SOARES, M.B. Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 1998. Não consta Alfabetização e letramento II Não consta SOARES, M. B. Letramento e Alfabetização: as muitas facetas. Rev. Brasileira Educação. Rio de Janeiro, n.25, 2004, p.5-14. SOARES, M.B. Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 1998 Ensino e aprendizagem de linguagem I SOARES M. B. Língua escrita, sociedade e cultura. Revista Brasileira de Educação. (0): 5-16. Belo Horizonte, ANPED, 1995. SOARES, M. B. Letramento e alfabetização: um tema em três gêneros. MG: Autêntica, 1999. SOARES, Magda Becker. Alfabetização: em busca de um método? Belo Horizonte: EDUC. REV., 1990. SOARES, Magda Becker. Letramento em verbete: o que é letramento? Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 2003. SOARES, Magda Becker. Paulo Freire - Alfabetização: muito além de um método. Revista Presença pedagógica. Belo Horizonte, n.21, maio/jun. 1998. SOARES, Magda. Alfabetização: a ressignificação do conceito. Revista Alfabetização Não indicado Ensino e aprendizagem de linguagem II SOARES, Magda Becker, (1998). Letramento: um tema em três gêneros. Belo Horizonte: Autêntica. SOARES, Magda Becker, MACIEL, Francisca, (2000). Alfabetização. Brasília: MEC/INEP/ COMPED (série Estado do Conhecimento). SOARES, Magda. Letramento: um tema em três gêneros. Autêntica Editora, 1998. SOARES, Magda. Letramento e alfabetização: as muitas facetas. 2000. Soares, Magda. Novas práticas de leitura e escrita: letramento na cibercultura. Educação e Sociedade 23.81 (2002): 143-160. Soares, Magda. Português na escola: história de uma disciplina curricular. Lingüística da norma. São Paulo: Edições Loyola (2002): 155-177. Soares, Magda. Português na escola: história de uma disciplina curricular. Lingüística da norma. São Paulo: Edições Loyola (2002): 155-177. 14 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 14 Revista Brasileira de Educação, n.º 25, p. 5-17, Jan./Abr. 2004. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 15 - 2023 UFVJM Alfabetização e Letramento SOARES, M. Alfabetização: a questão dos métodos. Contexto, 2016. SOARES, Magda. Letramento: um tema em três gêneros. 2ed. Belo Horizonte – MG: Autêntica, 2001. Alfabetização e Letramento I Não consta Não consta UFU Princípios e Métodos de Alfabetização SOARES, Magda. Letramento: um tema em três gêneros. Belo Horizonte: Autêntica, 2009. Não consta Processo de Alfabetização I Não consta Não consta Processo de Alfabetização II Não consta SOARES, M. Letramento: um tema em três gêneros. 3. ed. Belo Horizonte: Autêntica, 2009. UFTM Escrita, alfabetização e letramento SOARES, M. Alfabetização e letramento. São Paulo: Contexto 2008. Não consta Dentre as obras citadas de Magda Soares como base teórica dos componentes curriculares de alfabetização destacam-se artigos, livros e capítulos de livros em que a temática central se encontra no conceito ou na concepção de letramento. O termo letramento não foi criado por Magda Soares, nem mesmo a teoria a respeito do letra mento se inicia com a autora, mas as discussões se estruturam a partir de diferentes pesquisas de senvolvidas por ela. Em Soares (1985) “As muitas facetas da alfabetização” tem-se a discussão do conceito e da perspectiva das diferentes facetas que envolve o processo de alfabetização, ou seja, pensar a alfabetizar requer acionar conhecimentos de outros campos teóricos. Tal reflexão é retoma da em estudos publicados na XVII Reunião Anual da ANPED em 1995, e retomados de forma mais detalhada na obra “Letramento: um tema em três gêneros”, que teve sua primeira edição em 1998. Mais adiante, na obra “Alfabetização e letramento”, com a primeira edição em 2003, a autora retoma textos anteriormente publicados para rediscuti-los, aponta as diferentes perspectivas que constituem o processo de alfabetização, como as perspectivas histórica, antropológica, sociológica, psicológica e psicolinguística, sociolinguística, linguística, discursiva, textual, educacional ou pedagógica e políti ca. Considerações finais Esta pesquisa considera que a formação docente propõe ações que acionam diferentes conhe cimentos, organizados e transmitidos por instituições de ensino superior, de modo a assegurar a for mação de saberes científicos, pedagógicos, disciplinares, experienciais (TARDIF, 2014), compondo uma formação mais abrangente do profissional pedagogo. Diante disso, para a formação e a atuação de professores alfabetizadores, entende-se que tais conhecimentos exigem reflexões, apreensões e percepções teóricas e metodológicas mais aprofundadas frente às especificidades do processo de alfabetização. Diante disso, a pesquisa aponta que a fundamentação teórica acerca da alfabetização e letra mento aparece demarcada por componentes curriculares nos cursos de Pedagogia, com ênfase em aspectos conceituais e práticos em relação à alfabetização e ao letramento, com base nas obras de Magda Soares.l Ao refletir-se sobre as concepções que alicerçam a política de formação de professores, pode-se afirmar que a melhoria da educação e do ensino deve ultrapassar a implantação de diretrizes legais que privilegiam o aligeiramento e a perspectiva tecnicista da formação, muitas vezes descomprome tida com a pesquisa, a investigação e a formação multidisciplinar sólida; não privilegiar os processos de avaliação de desempenho e de competências vinculadas ao saber fazer e ao como fazer, ao invés de processos que tomam o campo da educação em sua totalidade. Dessa forma, observar a presença de autores de referência para a área da alfabetização, como a professora Magda Soares, inseridos nos estudos de componentes da área nos cursos de formação inicial, implica refletir sobre as concepções que vão alicerçar a construção dos saberes desses futuros profissionais. Destaca-se que “nenhum saber é por si mesmo formador” (TARDIF, 2014, p. 43), visto que ne nhum conhecimento transmitido garante por si mesmo a eficácia da formação docente. O que requer um posicionamento mais atuante, dialógico e reflexivo das ações formativas no contexto das institui ções de ensino superior, na articulação entre das dimensões teórico e prática, especificamente em relação a área da alfabetização. Como também uma reflexão redirecionada à dialogicidade como ato ativador do processo formativo, em ações de retomada das discussões, de construção, reconstrução ou desconstrução de diálogos interiores, de modo a garantir que os sujeitos se “formem e se consti tuam para, assim, contribuírem de modo significativo no processo de formação do outro” (GOULART, 2016, p. 723), e a perceber que “[...] educar incorpora as marcas de um ofício e de uma arte, apren dida no diálogo de gerações” (ARROYO, 2013, p.18). Na mesma vertente, o texto “Letramento e Alfabetização: as muitas facetas”, publicado em 2004, traz uma discussão mais refinada do conceito de alfabetização e letramento bem como assinala as interfaces do processo: parece ser necessário rever os quadros referenciais e os processos de ensino que têm predominado em nossas salas de aula, e talvez reconhecer a possibilidade e mesmo a necessidade de estabelecer a distinção entre o que mais propriamente se denomina le tramento, de que são muitas as facetas – imersão das crianças na cultura escrita, partici pação em experiências variadas com a leitura e a escrita, conhecimento e interação com diferentes tipos e gêneros de material escrito – e o que é propriamente a alfabetização, de que também são muitas as facetas – consciência fonológica e fonêmica, identificação das relações fonema–grafema, habilidades de codificação e decodificação da língua escrita, conhecimento e reconhecimento dos processos de tradução da forma sonora da fala para a forma gráfica da escrita (SOARES, 2004, p. 15). Frente à complexidade que envolve o processo de alfabetização e letramento, o qual abarca conhecimentos de áreas distintas, é necessário vislumbrar compreensões possíveis referentes às necessidades formativas do professor alfabetizador, que permita “[...] compreender todas as facetas (psicológica, psicolinguística, sociolinguística e linguística e todos os condicionantes (sociais, cultu rais, políticos) do processo de alfabetização” (SOARES, 2003, p. 24). Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 16 AZARIAS, Lívia de. S. T.; GOULART, I. do C. V.; CABRAL, G. R. Momentos formativos e fazer pedagógico nas palavras de professoras alfabetizadoras. Revista Eletrônica de Educação, [S. l.], v. 17, p. e4921014, 2023. ARROYO, Miguel. Ofício de mestre: imagens e autoimagens. 15. ed. Petrópolis: Vozes, 2013. BRASIL. Resolução CNE/CP 2/2019. Diário Oficial da União, Brasília, 15 de abril de 2020, Seção 1, pp. 46-49. BRASIL. BRASIL. Base Nacional Comum Curricular - Educação é a Base. Brasília, MEC/CONSED/UNDIME, 2018. BRASIL. Conselho Nacional de Educação. Resolução n.1, 15.5.2006. Diário Oficial da União, n.92, seção 1, p.11- 12, 16 maio 2006. CABRAL, Giovanna Rodrigues. Programa de formação continuada de professores: Pró Letramento em ação. Dissertação Mestrado em Educação – Universidade Católica de Petrópolis Centro de Teologia e Humanidade, Petrópolis, 2010. CABRAL, G. R.; GOULART, Ilsa do Carmo Vieira; NAVES, Ludmila Magalhães. Apresentação do dossiê. Devir Educação, Lavras, vol. 6, n.1, e–592, 2022. CABRAL, Giovanna Rodrigues; GOULART, Ilsa do Carmo Vieira; AQUINO, Kenia Adriano. Apresentação do dossiê: Ações e contextos formativos de professores alfabetizadores. Revista Brasileira de Alfabetização, n.17, 2022. CUNHA, Maria Isabel; ZANCHET, Beatriz Boéssio Atrib; RIBEIRO, Gabriela Machado. Qualidade do ensino de graduação: culturas, valores e seleção de professores. Práxis Educativa, Ponta Grossa, v. 8, n. 1, p. 219-241, jan./jun. 2013. GATTI, Bernardete. Formação de professores no Brasil: características e problemas. Revista Educação e Sociedade, Campinas, v. 31, n. 113, p. 1355-1379, out./dez. 2010. GATTI, Bernardete. Formação inicial de professores para a educação básica: pesquisas e políticas educacionais. Revista Estudos em Avaliação Educacional, São Paulo, v. 25, n. 57, p. 24-54, jan./abr. 2014. GOULART, Ilsa do Carmo Vieira Goulart. Linguagem, dialogicidade e docência: GOULART, Ilsa do Carmo Vieira Goulart. Linguagem, dialogicidade e docência: ormação em atos. Revista Diálogo Educacional, Curitiba, v. 16, n. 49, p. 705-726, jul./set. 2016. o processo de formação em atos. Revista Diálogo Educacional, Curitiba, v. 16, n. 49, p. 705-726 GOULART, Ilsa do Carmo Vieira; CABRAL, Giovanna Rodrigues. Apresentação do Dossiê Formação de Professores Alfabetizadores: políticas, saberes e práticas. Revista Eletrônica de Educação, [S. l.], v. 17, p. e6313002, 2023. IMBERNÓN, Francisco. Formação docente e profissional: formar-se para a mudança e a incerteza. 9. ed. São Paulo: Cortez, 2011. IMBERNÓN, Francisco. Ser docente em uma sociedade compleja: la difícil tarea de enseñar. Barcelona: Editorial GRAÓ, 2017. MARTINS FILHO, Lourival José; BARROS-MENDES, Adelma. das Neves Nunes; OLIVEIRA, Fernando. EDITORIAL. Revista Brasileira De Alfabetização, n. 16, 2022. NÓVOA, Antonio. (Coord.). Formação de professores e profissão docente. In: NÓVOA, Antonio. Os professores e a sua formação. Lisboa: Dom Quixote, 1992. p. 13-33. NÓVOA, Antonio. Firmar a profissão com o professor, afirmar a profissão docente. Cadernos de Pesquisa, São Paulo, v. 47, n. 166, p. 1106-1133, 2017. Referências ANDRÉ, Marli. A produção acadêmica sobre formação docente: um estudo comparativo das dissertações e teses dos anos 1990 e 2000. Revista Brasileira de Pesquisa sobre Formação de Professores, v. 1, n. 1, p. 41-56, ago./dez. 2009. ANDRÉ, Marli. Formação de professores: a constituição de um campo de estudos. Educação, PUC/ RS, v. 33, p. 6-18, 2010. ARROYO, Miguel. Ofício de mestre: imagens e autoimagens. 15. ed. Petrópolis: Vozes, 2013. ARROYO, Miguel. Ofício de mestre: imagens e autoimagens. 15. ed. Petrópolis: Vozes, 2013. AZARIAS, Lívia de. S. T.; GOULART, I. do C. V.; CABRAL, G. R. Momentos formativos e fazer pedagógico nas palavras de professoras alfabetizadoras. Revista Eletrônica de Educação, [S. l.], v. 17, p. e4921014, 2023. Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 17 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 17 Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 17 NÓVOA, Antonio. Para uma formação de professores construída dentro da profissão. In: NÓVOA, Antonio. Professores: Imagens do futuro presente. Lisboa: Educa: 2009, p.25-45. NÓVOA, Antonio. Os Professores e a sua Formação num Tempo de Metamorfose da Escola. Educação & Realidade, Porto Alegre, v. 44, n. 3, e84910, 2019. TA, Selma, Garrido. (org.). Saberes pedagógicos e atividade docente. São Paulo: Cortez, 1999. PINTO, E. G. R. P.; GOULART, I. C. V.; CABRAL, G. R. Formação de professores alfabetizadores: uma reflexão a partir das percepções do processo formativo no PNAIC. Revista Educação e Políticas em Debate, v. 10, n. 2, p. 544-560, mai./ago. 2021. 18 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 Revista Brasileira de Alfabetização \| ISSN: 2446-8584 \| Número 20 (Edição Especial) - 2023 18 SILVA, F. D. A.; SIGNORELLI, G. Apresentação - Alfabetização no Brasil: Olhares sobre a história, as políticas públicas e práticas educativas. Revista Educação e Políticas em Debate, [S. l.], v. 10, n. 2, p. 506-514, 2021. 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https://openalex.org/W2226044131	https://digital.library.adelaide.edu.au/dspace/bitstream/2440/104011/2/hdl_104011.pdf	English	null	Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry	Journal of Cosmology and Astroparticle Physics	2,016	cc-by	18,365	http://creativecommons.org/licenses/by/3.0/ http://creativecommons.org/licenses/by/3.0/ PUBLISHED VERSION M.G. Aarten ... G.C. Hill ... S. Robertson ... B.J. Whelan ... et al. M.G. Aarten ... G.C. Hill ... S. Robertson ... B.J. Whelan ... et al. Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry Journal of Cosmology and Astroparticle Physics 2016; 2016(4):022 1 022 25 p p y y Journal of Cosmology and Astroparticle Physics, 2016; 2016(4):022-1-022-25 Article funded by SCOAP. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 License. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Originally published at: http://doi.org/10.1088/1475-7516/2016/04/022 http://hdl.handle.net/2440/104011 http://hdl.handle.net/2440/104011 Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry ournal of Cosmology and Astroparticle Physics An IOP and SISSA journal J ournal of Cosmology and Astroparticle Physics An IOP and SISSA journal J Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry Use of event-level neutrino telescope data in global fits for theories of new physics P. Scott, C. Savage, J. Edsjö et al. Limits on dark matter proton scattering from neutrino telescopes using micrOMEGAs G. Bélanger, J. Da Silva, T. Perrillat-Bottonet et al. Generalised form factor dark matter in the Sun Aaron C. Vincent, Aldo Serenelli and Pat Scott IceCube, DeepCore, PINGU and the indirect search for supersymmetric dark matter Paul Bergeron and Stefano Profumo AMS-02 and LHC Abdesslam Arhrib, Yue-Lin Sming Tsai, Qiang Yuan et al. Updated constraints on velocity and momentum-dependent asymmetric dark matter Aaron C. Vincent, Pat Scott and Aldo Serenelli WIMPs at the galactic center Prateek Agrawal, Brian Batell, Patrick J. Fox et al. Global fits of the dark matter-nucleon effective interactions Riccardo Catena and Paolo Gondolo Direct and indirect detection of dissipative dark matter JiJi Fan, Andrey Katz and Jessie Shelton Updated constraints on velocity and momentum-dependent asymmetric dark matter Aaron C. Vincent, Pat Scott and Aldo Serenelli WIMPs at the galactic center Prateek Agrawal, Brian Batell, Patrick J. Fox et al. Global fits of the dark matter-nucleon effective interactions Riccardo Catena and Paolo Gondolo Direct and indirect detection of dissipative dark matter JiJi Fan, Andrey Katz and Jessie Shelton Direct and indirect detection of dissipative dark matter JiJi Fan, Andrey Katz and Jessie Shelton Article funded by SCOAP3. Content from this work may be used under the terms of the Creative Commons Attribution 3.0 License. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry Mase,15 H.S. Matis,8 R. Maunu,17 F. McNally,30 K. Meagher,12 M. Medici,20 M. Meier,21 A. Meli,26 T. Menne,21 G. Merino,30 T. Meures,12 S. Miarecki,8,7 E. Middell,50 L. Mohrmann,50 T. Montaruli,25 R. Morse,30 R. Nahnhauer,50 U. Naumann,49 G. Neer,22 H. Niederhausen,41 S.C. Nowicki,23 D.R. Nygren,8 A. Obertacke Pollmann,49 A. Olivas,17 A. Omairat,49 A. O’Murchadha,12 T. Palczewski,44 H. Pandya,34 D.V. Pankova,46 L. Paul,1 J.A. Pepper,44 C. P´erez de los Heros,48 C. Pfendner,18 D. Pieloth,21 E. Pinat,12 J. Posselt,49 P.B. Price,7 G.T. Przybylski,8 M. Quinnan,46 C. Raab,12 L. R¨adel,1 M. Rameez,25 K. Rawlins,3 R. Reimann,1 M. Relich,15 E. Resconi,33 W. Rhode,21 M. Richman,37 S. Richter,30 B. Riedel,23 S. Robertson,2 M. Rongen,1 C. Rott,42 T. Ruhe,21 D. Ryckbosch,26 L. Sabbatini,30 H.-G. Sander,31 A. Sandrock,21 J. Sandroos,31 S. Sarkar,20,36 C. Savage,40 K. Schatto,31 M. Schimp,1 P. Schlunder,21 T. Schmidt,17 S. Schoenen,1 S. Sch¨oneberg,10 A. Sch¨onwald,50 L. Schulte,11 L. Schumacher,1 P. Scott,d,f D. Seckel,34 S. Seunarine,39 H. Silverwood,16,e D. Soldin,49 M. Song,17 G.M. Spiczak,39 C. Spiering,50 P.O. Hulth,40 K. Hultqvist,40 S. In,42 A. Ishihara,15 E. Jacobi, P.O. Hulth,40 K. Hultqvist,40 S. In,42 A. Ishihara,15 E. Jacobi,50 G.S. Japaridze,4 M. Jeong,42 K. Jero,30 B.J.P. Jones,14 p g M. Jurkovic,33 A. Kappes,24 T. Karg,50 A. Karle,30 U. Katz,24 30 35 46 30 1 M. Kauer,30,35 A. Keivani,46 J.L. Kelley,30 J. Kemp,1 y p A. Kheirandish,30 J. Kiryluk,41 S.R. Klein,8,7 G. Kohnen,32 R. Koirala,34 H. Kolanoski,9 R. Konietz,1 L. K¨opke,31 C. Kopper 49 20 9 50 33 . Kopper,49 D.J. Koskinen,20 M. Kowalski,9,50 K. Krings,33 pp g G. Kroll,31 M. Kroll,10 G. Kr¨uckl,31 J. Kunnen,13 N. Kurahashi,37 G. Kroll,31 M. Kroll,10 G. Kr¨uckl,31 J. Kunnen,13 N. Kurahashi,37 T K b 15 M L b 26 J L L f hi 46 M J L 20 T. Kuwabara,15 M. Labare,26 J.L. Lanfranchi,46 M.J. Larson,20 M. Lesiak-Bzdak,41 M. Leuermann,1 J. Leuner,1 L. Lu,15 gg Mandelartz,10 R. Maruyama,35 K. Mase,15 H.S. Matis,8 . Maunu,17 F. McNally,30 K. Meagher,12 M. Medici,20 M. Meier, . Meli,26 T. Menne,21 G. Merino,30 T. Meures,12 S. Miarecki,8,7 . Middell,50 L. Mohrmann,50 T. Montaruli,25 R. Morse,30 . Nahnhauer,50 U. Naumann,49 G. Neer,22 H. Niederhausen,41 23 8 49 .C. Nowicki,23 D.R. Nygren,8 A. Obertacke Pollmann,49 . Olivas,17 A. Omairat,49 A. O’Murchadha,12 T. Palczewski,44 H. Pandya,34 D.V. Pankova,46 L. Paul,1 J.A. Pepper,44 y pp C. P´erez de los Heros,48 C. Pfendner,18 D. Pieloth,21 E. Pinat,12 49 7 8 46 J. Posselt,49 P.B. Price,7 G.T. Przybylski,8 M. Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry JCAP04(2016)022 The IceCube collaboration M.G. Aartsen,2 K. Abraham,33 M. Ackermann,50 J. Adams,16 J.A. Aguilar,12 M. Ahlers,30 M. Ahrens,40 D. Altmann,24 T. Anderson,46 I. Ansseau,12 G. Anton,24 M. Archinger,31 C. Arguelles,14 T.C. Arlen,46 J. Auﬀenberg,1 X. Bai,38 S.W. Barwick,27 V. Baum,31 R. Bay,7 J.J. Beatty,18,19 J. Becker Tjus,10 K.-H. Becker,49 E. Beiser,30 S. BenZvi,47 P. Berghaus,50 D. Berley,17 E. Bernardini,50 A. Bernhard,33 D.Z. Besson,28 G. Binder,8,7 D. Bindig,49 M. Bissok,1 E. Blaufuss,17 J. Blumenthal,1 D.J. Boersma,48 C. Bohm,40 M. B¨orner,21 F. Bos,10 D. Bose,42 S. B¨oser,31 O. Botner,48 J. Braun,30 L. Brayeur,13 H.-P. Bretz,50 N. Buzinsky,23 J. Casey,5 M. Casier,13 E. Cheung,17 D. Chirkin,30 A. Christov,25 K. Clark,43 L. Classen,24 S. Coenders,33 G.H. Collin,14 J.M. Conrad,14 D.F. Cowen,46,45 A.H. Cruz Silva,50 M. Danninger,40,c,f J. Daughhetee,5 J.C. Davis,18 M. Day,30 J.P.A.M. de Andr´e,22 C. De Clercq,13 E. del Pino Rosendo,31 H. Dembinski,34 S. De Ridder,26 P. Desiati,30 K. D. de Vries,13 G. de Wasseige,13 M. de With,9 T. DeYoung,22 J.C. D´ıaz-V´elez,30 V. di Lorenzo,31 J.P. Dumm,40 M. Dunkman,46 B. Eberhardt,31 J. Edsj¨o,40 T. Ehrhardt,31 B. Eichmann,10 S. Euler,48 P.A. Evenson,34 S. Fahey,30 A.R. Fazely,6 J. Feintzeig,30 J. Felde,17 K. Filimonov,7 C. Finley,40 S. Flis,40 C.-C. F¨osig,31 T. Fuchs,21 The IceCube collaboration . Berghaus,50 D. Berley,17 E. Bernardini,50 A. Bernhard,33 28 8 7 49 1 D.Z. Besson,28 G. Binder,8,7 D. Bindig,49 M. Bissok,1 E. Blaufuss,1 . Blumenthal,1 D.J. Boersma,48 C. Bohm,40 M. B¨orner,21 F. Bos, D. Bose,42 S. B¨oser,31 O. Botner,48 J. Braun,30 L. Brayeur,13 P.A. Evenson,34 S. Fahey,30 A.R. Fazely,6 J. Feintzeig,30 J. Felde,17 K. Filimonov,7 C. Finley,40 S. Flis,40 C.-C. F¨osig,31 T. Fuchs,21 doi:10.1088/1475-7516/2016/04/022 JCAP04(2016)022 T.K. Gaisser,34 R. Gaior,15 J. Gallagher,29 L. Gerhardt,8,7 K. Ghorbani,30 D. Gier,1 L. Gladstone,30 M. Glagla,1 T. Gl¨usenkamp,50 A. Goldschmidt,8 G. Golup,13 J.G. Gonzalez,34 D. G´ora,50 D. Grant,23 Z. Griﬃth,30 A. Groß,33 C. Ha,8,7 C. Haack,1 A. Haj Ismail,26 A. Hallgren,48 F. Halzen,30 E. Hansen,20 B. Hansmann,1 K. Hanson,30 D. Hebecker,9 D. Heereman,12 K. Helbing,49 R. Hellauer,17 S. Hickford,49 J. Hignight,22 G.C. Hill,2 K.D. Hoﬀman,17 R. Hoﬀmann,49 K. Holzapfel,33 A. Homeier,11 K. Hoshina,30,a F. Huang,46 M. Huber,33 W. Huelsnitz,17 P.O. Hulth,40 K. Hultqvist,40 S. In,42 A. Ishihara,15 E. Jacobi,50 G.S. Japaridze,4 M. Jeong,42 K. Jero,30 B.J.P. Jones,14 M. Jurkovic,33 A. Kappes,24 T. Karg,50 A. Karle,30 U. Katz,24 M. Kauer,30,35 A. Keivani,46 J.L. Kelley,30 J. Kemp,1 A. Kheirandish,30 J. Kiryluk,41 S.R. Klein,8,7 G. Kohnen,32 R. Koirala,34 H. Kolanoski,9 R. Konietz,1 L. K¨opke,31 C. Kopper,23 S. Kopper,49 D.J. Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry Koskinen,20 M. Kowalski,9,50 K. Krings,33 G. Kroll,31 M. Kroll,10 G. Kr¨uckl,31 J. Kunnen,13 N. Kurahashi,37 T. Kuwabara,15 M. Labare,26 J.L. Lanfranchi,46 M.J. Larson,20 M. Lesiak-Bzdak,41 M. Leuermann,1 J. Leuner,1 L. Lu,15 J. L¨unemann,13 J. Madsen,39 G. Maggi,13 K.B.M. Mahn,22 M. Mandelartz,10 R. Maruyama,35 K. Mase,15 H.S. Matis,8 R. Maunu,17 F. McNally,30 K. Meagher,12 M. Medici,20 M. Meier,21 A. Meli,26 T. Menne,21 G. Merino,30 T. Meures,12 S. Miarecki,8,7 E. Middell,50 L. Mohrmann,50 T. Montaruli,25 R. Morse,30 R. Nahnhauer,50 U. Naumann,49 G. Neer,22 H. Niederhausen,41 S.C. Nowicki,23 D.R. Nygren,8 A. Obertacke Pollmann,49 A. Olivas,17 A. Omairat,49 A. O’Murchadha,12 T. Palczewski,44 H. Pandya,34 D.V. Pankova,46 L. Paul,1 J.A. Pepper,44 C. P´erez de los Heros,48 C. Pfendner,18 D. Pieloth,21 E. Pinat,12 J. Posselt,49 P.B. Price,7 G.T. Przybylski,8 M. Quinnan,46 C. Raab,12 L. R¨adel,1 M. Rameez,25 K. Rawlins,3 R. Reimann,1 M. Relich,15 E. Resconi,33 W. Rhode,21 M. Richman,37 S. Richter,30 B. Riedel,23 S. Robertson,2 M. Rongen,1 C. Rott,42 T. Ruhe,21 D. Ryckbosch,26 L. Sabbatini,30 H.-G. Sander,31 A. Sandrock,21 J. Sandroos,31 S. Sarkar,20,36 C. Savage,40 K. Schatto,31 M. Schimp,1 P. Schlunder,21 T. Schmidt,17 S. Schoenen,1 S. Sch¨oneberg,10 A. Sch¨onwald,50 L. Schulte,11 L. Schumacher,1 P. Scott,d,f D. Seckel,34 S. Seunarine,39 H. Silverwood,16,e D. Soldin,49 M. Song,17 G.M. Spiczak,39 C. Spiering,50 T.K. Gaisser,34 R. Gaior,15 J. Gallagher,29 L. Gerhardt,8,7 K. Ghorbani,30 D. Gier,1 L. Gladstone,30 M. Glagla,1 g T. Gl¨usenkamp,50 A. Goldschmidt,8 G. Golup,13 J.G. Gonzalez,34 D. G´ora,50 D. Grant,23 Z. Griﬃth,30 A. Groß,33 C. Ha,8,7 C. Haac A. Haj Ismail,26 A. Hallgren,48 F. Halzen,30 E. Hansen,20 . Hansmann,1 K. Hanson,30 D. Hebecker,9 D. Heereman,12 K. Helbing,49 R. Hellauer,17 S. Hickford,49 J. Hignight,22 G.C. Hill g g g K.D. Hoﬀman,17 R. Hoﬀmann,49 K. Holzapfel,33 A. Homeier,11 K. Hoshina,30,a F. Huang,46 M. Huber,33 W. Huelsnitz,17 JCAP04(2016)022 JCAP04(2016)022 K. Hoshina,30, F. Huang, 6 M. Huber,33 W. Huelsnitz, 7 P.O. Hulth,40 K. Hultqvist,40 S. In,42 A. Ishihara,15 E. Jacobi,50 G.S. Japaridze,4 M. Jeong,42 K. Jero,30 B.J.P. Jones,14 M. Jurkovic,33 A. Kappes,24 T. Karg,50 A. Karle,30 U. Katz,24 M. Kauer,30,35 A. Keivani,46 J.L. Kelley,30 J. Kemp,1 A. Kheirandish,30 J. Kiryluk,41 S.R. Klein,8,7 G. Kohnen,32 R. Koirala,34 H. Kolanoski,9 R. Konietz,1 L. K¨opke,31 C. Kopper,23 S. Kopper,49 D.J. Koskinen,20 M. Kowalski,9,50 K. Krings,33 G. Kroll,31 M. Kroll,10 G. Kr¨uckl,31 J. Kunnen,13 N. Kurahashi,37 T. Kuwabara,15 M. Labare,26 J.L. Lanfranchi,46 M.J. Larson,20 M. Lesiak-Bzdak,41 M. Leuermann,1 J. Leuner,1 L. Lu,15 J. L¨unemann,13 J. Madsen,39 G. Maggi,13 K.B.M. Mahn,22 M. Mandelartz,10 R. Maruyama,35 K. Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry Quinnan,46 12 1 25 3 C. Raab,12 L. R¨adel,1 M. Rameez,25 K. Rawlins,3 R. Reimann,1 M. Relich,15 E. Resconi,33 W. Rhode,21 M. Richman,37 S. Richter,3 23 2 1 42 21 . Riedel,23 S. Robertson,2 M. Rongen,1 C. Rott,42 T. Ruhe,21 D. Ryckbosch,26 L. Sabbatini,30 H.-G. Sander,31 A. Sandrock,21 . Sandroos,31 S. Sarkar,20,36 C. Savage,40 K. Schatto,31 g M. Schimp,1 P. Schlunder,21 T. Schmidt,17 S. Schoenen,1 . Sch¨oneberg,10 A. Sch¨onwald,50 L. Schulte,11 L. Schumacher,1 d f 34 39 16 . Scott,d,f D. Seckel,34 S. Seunarine,39 H. Silverwood,16,e D. Soldin,49 M. Song,17 G.M. Spiczak,39 C. Spiering,50 M. Stahlberg,1 M. Stamatikos,18,b T. Stanev,34 A. Stasik,50 g A. Steuer,31 T. Stezelberger,8 R.G. Stokstad,8 A. St¨oßl,50 g R. Str¨om,48 N.L. Strotjohann,50 G.W. Sullivan,17 M. Sutherland j H. Taavola,48 I. Taboada,5 J. Tatar,8,7 S. Ter-Antonyan,6 y A. Terliuk,50 G. Teˇsi´c,46 S. Tilav,34 P.A. Toale,44 M.N. Tobin,30 4 S. Toscano,13 D. Tosi,30 M. Tselengidou,24 A. Turcati,33 g E. Unger,48 M. Usner,50 S. Vallecorsa,25 J. Vandenbroucke,30 N. van Eijndhoven,13 S. Vanheule,26 J. van Santen,50 j J. Veenkamp,33 M. Vehring,1 M. Voge,11 M. Vraeghe,26 JCAP04(2016)022 p g g g C. Walck,40 A. Wallace,2 M. Wallraﬀ,1 N. Wandkowsky,30 Ch. Weaver,23 C. Wendt,30 S. Westerhoﬀ,30 B.J. Whelan,2 44 K. Wiebe,31 C.H. Wiebusch,1 L. Wille,30 D.R. Williams,44 L. Wills,37 H. Wissing,17 M. Wolf,40 T.R. Wood,23 K. Wosch D.L. Xu,30 X.W. Xu,6 Y. Xu,41 J.P. Yanez,50 G. Yodh,27 S Yoshida15 and M Zoll40 K. Wiebe,31 C.H. Wiebusch,1 L. Wille,30 D.R. Williams,44 7 7 4 K. Wiebe,31 C.H. Wiebusch,1 L. Wille,30 D.R. Williams,44 L. Wills,37 H. Wissing,17 M. Wolf,40 T.R. Wood,23 K. Wosc D L X 30 X W X 6 Y X 41 J P Y 50 G Y dh 27 L. Wills,37 H. Wissing,17 M. Wolf,40 T.R. Wood,23 K. Woschnag L. Wills,37 H. Wissing,17 M. Wolf,40 T.R. Wood,23 K. Woschnagg,7 D.L. Xu,30 X.W. Xu,6 Y. Xu,41 J.P. Yanez,50 G. Yodh,27 S. Yoshida15 and M. Zoll40 g D.L. Xu,30 X.W. Xu,6 Y. Xu,41 J.P. Yanez,50 G. Yodh,27 S Y hid 15 d M Z ll40 1III. Physikalisches Institut, RWTH Aachen University, Aachen, D-52056 Germany 1III. Physikalisches Institut, RWTH Aachen University, A Aachen, D-52056 Germany 2Department of Physics, University of Adelaide, Adelaide, 5005 Australia 2Department of Physics, University of Adelaide, Adelaide, 5005 Australia 3Dept. of Physics and Astronomy, University of Alaska Anchorage, 3211 Providence Dr., Anchorage, AK, 99508 U.S.A. 3Dept. 12Universit´e Libre de Bruxelles, Science Faculty CP230, Brussels, B-1050 Belgium 12Universit´e Libre de Bruxelles, Science Faculty CP230, Brussels, B-1050 Belgium Improved limits on dark matter annihilation in the Sun with the 79-string IceCube detector and implications for supersymmetry of Physics and Astronomy, University of Alaska Anchorage, 3211 Providence Dr., Anchorage, AK, 99508 U.S.A. 3Dept. of Physics and Astronomy, University of Alaska Anchorage, 3Dept. of Physics and Astronomy, University of Alask 3211 Providence Dr., Anchorage, AK, 99508 U.S.A. 3211 Providence Dr., Anchorage, AK, 99508 U.S.A. 4CTSPS, Clark-Atlanta University, Atlanta, GA, 30314 U.S.A. 4CTSPS, Clark-Atlanta University, Atlanta, GA, 30314 U.S.A. 4CTSPS, Clark-Atlanta University, Atlanta, GA, 30314 U.S.A. 5School of Physics and Center for Relativistic Astrophysics, Georgia Institute of Technology, Atlanta, GA, 30332 U.S.A. 5School of Physics and Center for Relativistic Astrophysics, Georgia Institute of Technology, Atlanta, GA, 30332 U.S.A. 6Dept. of Physics, Southern University, 6Dept. of Physics, Southern University, Baton Rouge, LA, 70813 U.S.A. Baton Rouge, LA, 70813 U.S.A. 7Dept. of Physics, University of California, 7Dept. of Physics, University of California, Berkeley, CA, 94720 U.S.A. 8Lawrence Berkeley National Laboratory, 8Lawrence Berkeley National Laboratory, Berkeley, CA, 94720 U.S.A. 9Institut f¨ur Physik, Humboldt-Universit¨at zu Berlin, Berlin, D-12489 Germany 9Institut f¨ur Physik, Humboldt-Universit¨at zu Berlin, Berlin, D-12489 Germany 10Fakult¨at f¨ur Physik & Astronomie, Ruhr-Universit¨at Bochum, Bochum, D-44780 Germany 10Fakult¨at f¨ur Physik & Astronomie, Ruhr-Universit¨at Bochum, Bochum, D-44780 Germany 11Physikalisches Institut, Universit¨at Bonn, N ll 12 B D 53115 G 11Physikalisches Institut, Universit¨at Bonn, Nussallee 12, Bonn, D-53115 Germany Nussallee 12, Bonn, D-53115 Germany Brussels, B-1050 Belgium 13Vrije Universiteit Brussel, Dienst ELEM, Brussels, B-1050 Belgium 14Dept. of Physics, Massachusetts Institute of Technology, Cambridge, MA, 02139 U.S.A. 15Dept. of Physics, Chiba University, Chiba, 263-8522 Japan 16Dept. of Physics and Astronomy, University of Canterbury, Private Bag 4800, Christchurch, New Zealand 16Dept. of Physics and Astronomy, University of Can Private Bag 4800, Christchurch, New Zealand 16Dept. of Physics and Astronomy, University of Canterbury, 17Dept. of Physics, University of Maryland, College Park, MD, 20742 U.S.A. 17Dept. of Physics, University of Maryland, College Park, MD, 20742 U.S.A. 18Dept. of Physics and Center for Cosmology and Astro-Particle Physics, Ohio State University, Columbus, OH, 43210 U.S.A. 19Dept. of Astronomy, Ohio State University, Columbus, OH, 43210 U.S.A. 19Dept. of Astronomy, Ohio State University, Columbus, OH, 43210 U.S.A. JCAP04(2016)022 JCAP04(2016)022 20Niels Bohr Institute, University of Copenhagen, Copenhagen, DK-2100 Denmark 20Niels Bohr Institute, University of Copenhagen, Copenhagen, DK-2100 Denmark 21Dept. of Physics, TU Dortmund University, Dortmund, D-44221 Germany 22Dept. of Physics and Astronomy, Michigan State University, East Lansing, MI, 48824 U.S.A. 22Dept. of Physics and Astronomy, Michigan State University, East Lansing, MI, 48824 U.S.A. 23Dept. of Physics, University of Alberta, Edmonton, Alberta, T6G 2E1 Canada 23Dept. of Physics, University of Alberta, Edmonton, Alberta, T6G 2E1 Canada 24Erlangen Centre for Astroparticle Physics, Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg, Erlangen, D-91058 Germany 24Erlangen Centre for Astroparticle Physics, Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg, Erlangen, D-91058 Germany 24Erlangen Centre for Astroparticle Physics, Friedrich-Alexander-Universit¨at Erlangen-N¨urnberg, Erlangen, D-91058 Germany Erlangen, D-91058 Germany 25D´epartement de physique nucl´eaire et corpusculaire, Universit´e de Gen`eve, Gen`eve, CH-1211 Switzerland 25D´epartement de physique nucl´eaire et corpusculaire, Universit´e de Gen`eve, Gen`eve, CH-1211 Switzerland 26Dept. of Physics and Astronomy, University of Gent, Gent, B-9000 Belgium 26Dept. of Physics and Astronomy, University of Gent, Gent, B-9000 Belgium 27Dept. of Physics and Astronomy, University of California, Irvine, CA, 92697 U.S.A. 28Dept. of Physics and Astronomy, University of Kansas, Lawrence, KS, 66045 U.S.A. 28Dept. of Physics and Astronomy, University of Kansas, Lawrence, KS, 66045 U.S.A. 28Dept. of Physics and Astronomy, University of Kansas, Lawrence, KS, 66045 U.S.A. 29Dept. of Astronomy, University of Wisconsin, Madison, WI, 53706 U.S.A. 29Dept. of Astronomy, University of Wisconsin, Madison, WI, 53706 U.S.A. 29Dept. of Astronomy, University of Wisconsin, Madison, WI, 53706 U.S.A. 30Dept. of Physics and Wisconsin IceCube Particle Astrophysics Center, University of Wisconsin, Madison, WI, 53706 U.S.A. 31Institute of Physics, University of Mainz, Staudinger Weg 7, Mainz, D-55099 German 32Universit´e de Mons, Mons, 7000 Belgium 32Universit´e de Mons, Mons, 7000 Belgium 33Technische Universit¨at M¨unchen, Garching, D-85748 Germany 33Technische Universit¨at M¨unchen, Garching, D-85748 Germany 34Bartol Research Institute and Dept. of Physics and Astronomy, University of Delaware, Newark, DE, 19716 U.S.A. 35Dept. of Physics, Yale University, New Haven, CT, 06520 U.S.A. 35Dept. of Physics, Yale University, New Haven, CT, 06520 U.S.A. 36Dept. of Physics, University of Oxford, 1 Keble Road, Oxford, OX1 3NP U.K. 36Dept. of Physics, University of Oxford, 1 Keble Road, Oxford, OX1 3NP U.K. 37Dept. of Physics, Drexel University, 3141 Chestnut Street, Philadelphia, PA, 19104 U.S.A. 37Dept. of Physics, Drexel University, 3141 Chestnut Street, Philadelphia, PA, 19104 U.S.A. 38Physics Department, South Dakota School of Mines and Technology, Rapid City, SD, 57701 U.S.A. 39Dept. of Physics, University of Wisconsin, River Falls, WI, 54022 U.S.A. 39Dept. of Physics, University of Wisconsin, River Falls, WI, 54022 U.S.A. 40Oskar Klein Centre and Dept. of Physics, Stockholm University, Stockholm, SE-10691 Sweden 41Dept. of Physics and Astronomy, Stony Brook University, Stony Brook, NY, 11794-3800 U.S.A. 41Dept. of Physics and Astronomy, Stony Brook University, Stony Brook, NY, 11794-3800 U.S.A. JCAP04(2016)022 JCAP04(2016)022 42Dept. of Physics, Sungkyunkwan University, Suwon, 440-746 Korea 42Dept. of Physics, Sungkyunkwan University, Suwon, 440-746 Korea 43Dept. of Physics, University of Toronto, Toronto, Ontario, M5S 1A7 Canada 43Dept. of Physics, University of Toronto, Toronto, Ontario, M5S 1A7 Canada 44Dept. of Physics and Astronomy, University of Alabama, Tuscaloosa, AL, 35487 U.S.A. 45Dept. Received January 6, 2016 Accepted March 24, 2016 Published April 12, 2016 Erlangen, D-91058 Germany Keywords: dark matter experiments, dark matter theory, neutrino experiments JCAP04(2016)022 ArXiv ePrint: 1601.00653 ArXiv ePrint: 1601.00653 Contents 1 Introduction 1 2 The 79-string IceCube search for dark matter 2 2.1 The IceCube detector 2 2.2 Data samples 3 2.3 Signal and background simulation 3 2.4 Calculation of detector eﬃciencies 3 2.5 Angular response 4 2.6 Energy estimator 4 2.7 Background estimation 5 2.8 Data format, public code and availability 5 3 Likelihood functions 9 3.1 General form 9 3.2 Background likelihood 10 3.3 Signal likelihood 10 3.4 Detector response 12 3.5 Predicted event rate 13 3.6 Bias factor calculation 13 4 Improved limits on WIMP dark matter 14 5 Implications for MSSM benchmarks 19 6 Conclusions 22 JCAP04(2016)022 4 Improved limits on WIMP dark matter 5 Implications for MSSM benchmarks 6 Conclusions Erlangen, D-91058 Germany of Astronomy and Astrophysics, Pennsylvania State University, University Park, PA, 16802 U.S.A. 46Dept. of Physics, Pennsylvania State University, University Park, PA, 16802 U.S.A. 47Dept. of Physics and Astronomy, University of Rochester, Rochester, NY, 14627 U.S.A. 48Dept. of Physics and Astronomy, Upp Box 516, Uppsala, S-75120 Sweden 48Dept. of Physics and Astronomy, Uppsala University, Box 516, Uppsala, S-75120 Sweden 48Dept. of Physics and Astronomy, Uppsala University, 49Dept. of Physics, University of Wuppertal, Wuppertal, D-42119 Germany pp , y 50DESY, Zeuthen, D-15735 Germany Zeuthen, D-15735 Germany aEarthquake Research Institute, University of Tokyo, Bunkyo, Tokyo, 113-0032 Japan bNASA Goddard Space Flight Center, Greenbelt, MD, 20771 U.S.A. cCurrent address: Dept. of Physics and Astronomy, University of British Columbia, Vancouver, V6T 1Z1 Canada dDept. of Physics, Imperial College London, London, SW7 2AZ U.K. eCurrent address: GRAPPA Institute, University of Amsterdam, Amsterdam, 1098 XH Netherlands fCorresponding authors: P. Scott and M. Danninger E-mail: matthias.danninger@cern.ch, p.scott@imperial.ac.uk Received January 6, 2016 Accepted March 24, 2016 Published April 12, 2016 aEarthquake Research Institute, University of Tokyo, Bunkyo, Tokyo, 113-0032 Japan bNASA Goddard Space Flight Center, Greenbelt, MD, 20771 U.S.A. cCurrent address: Dept. of Physics and Astronomy, University of British Columbia, Vancouver, V6T 1Z1 Canada eCurrent address: GRAPPA Institute, University of Amsterdam, Amsterdam, 1098 XH Netherlands fCorresponding authors: P. Scott and M. Danninger E-mail: matthias.danninger@cern.ch, p.scott@imperial.ac.uk E-mail: matthias.danninger@cern.ch, p.scott@imperial.ac.uk Abstract. We present an improved event-level likelihood formalism for including neutrino telescope data in global ﬁts to new physics. We derive limits on spin-dependent dark matter- proton scattering by employing the new formalism in a re-analysis of data from the 79-string IceCube search for dark matter annihilation in the Sun, including explicit energy information for each event. The new analysis excludes a number of models in the weak-scale minimal supersymmetric standard model (MSSM) for the ﬁrst time. This work is accompanied by the public release of the 79-string IceCube data, as well as an associated computer code for applying the new likelihood to arbitrary dark matter models. 1 Introduction Searches for high-energy neutrinos from the Sun are currently the most sensitive means of probing spin-dependent interactions between protons and most models for dark matter (DM) [1, 2]. Most analyses take a semi-model-independent approach, assuming that capture and annihilation have reached equilibrium in the Sun, and that DM annihilates exclusively into a single ﬁnal state. These assumptions are expressly violated in many concrete models for the identity of DM, including supersymmetry [3–7]. Resulting limits are often diﬃcult to meaningfully connect to theoretical predictions [8–15], in part because the necessary data and likelihood functions for recasting limits to other theories are unavailable. The compu- tational expense required to replicate the experimental analyses for millions of parameter combinations can also be prohibitive. All these issues arise in some form in direct detection, collider searches and other forms of indirect detection as well [16–21]. This paper provides a solution to these problems for the indirect dark matter search with neutrinos. We previously presented a 79-string search for dark matter annihilation in the Sun (IC79; [1]), deriving limits on single annihilation channels. We later developed a formalism (Paper I; [7]) that allows event-level neutrino telescope data to be used to constrain DM models with mixed annihilation ﬁnal states, thereby allowing IceCube searches to be properly – 1 – included in global ﬁts to theories beyond the Standard Model of particle physics (BSM). Paper I provided methods applicable to neutrinos with high energies (50 GeV and above) that were observed with the 22-string conﬁguration of IceCube. This paper (Paper II) revises this formalism to include the impact of non-negligible angles between the neutrino direction and the muon produced, extending the reach of the technique to neutrino energies as low as 10 GeV. We then apply the formalism to IC79 data and use it to rule out some example supersymmetric models. Compared to the original IC79 analysis [1, 22], which was based solely on the observed arrival directions of events, here we also include event-level energy information and an explicit treatment of the total number of observed events within the signal region, leading to an improvement in limits at high DM masses. Extensive references on neutrino searches for dark matter and BSM global ﬁts can be found in Paper I. 1 Introduction JCAP04(2016)022 We publicly provide the fast likelihood code (nulike1) that implements the improved analysis presented in this paper, using the public IC79 event information and detector re- sponse. Nulike also provides pre-computed, fully model-independent ‘partial likelihoods’ for every event observed by IC79, making new limits quick and easy to obtain for any annihilation ﬁnal state or combination thereof. This is a distinct advantage over the standard IceCube analysis pathway, where full signal propagation and detector simulations are required for each model. While the approach in this paper relies on many results of the direct simulation method, such as eﬀective areas and volumes, it provides a complete framework in which they can then be applied to essentially any neutrino annihilation signal that can be safely treated as a point source. The methods and the corresponding code are agnostic with respect to the details of the experiment and can be used to perform similar analyses for other neutrino telescopes, given appropriate input data in the form of event and detector response ﬁles. In section 2, we will provide details of the IC79 data that we use in the updated analysis, before describing the improved likelihood formalism in section 3. We then show the impacts of the new analysis on generic weakly-interacting massive particle (WIMP) models in section 4 and models in the minimal supersymmetric standard model (MSSM) in section 5. We will conclude in section 6. 1http://nulike.hepforge.org. Data samples 2.2 In the analysis described in this paper, we start with pre-selected data from a search for WIMP dark matter annihilation in the Sun with the IceCube 79-string conﬁguration [1]. This analysis uses 317 live-days of data taken between May 2010 and May 2011. As described in refs. [1, 22], the DeepCore subarray is included for the ﬁrst time in the analysis, lowering the energy threshold and extending the search to the austral summer (when neutrinos from the Sun pass downwards through the ice). In order to be sensitive to a wide range of potential WIMP masses, the analysis comprises three independent non-overlapping event selections. First, the full dataset is split into two seasonal streams, where September 22nd 2010 and March 22nd 2011 mark the beginning and end of the ‘summer’ dataset. The ‘summer’ sample (‘summer low-energy’ event selection, SL) is a dedicated low energy event sample that uses the surrounding IceCube strings as an instrumented muon veto in order to select neutrino- induced events that start within DeepCore. The ‘winter’ dataset comprises two samples. The ﬁrst sample (‘winter high-energy’ event selection, WH) has no particular track-containment requirement and aims to select upward-going muon tracks. The second sample (‘winter low- energy’ event selection, WL) is a low energy sample, and focuses on neutrino-induced muon tracks that start or are fully contained in DeepCore. The event selection was carried out separately for each independent sample. By design, the uncorrelated nature of the three datasets makes it straightforward to combine them in a joint likelihood. The analysis in sections 4 and 5 uses the event-level data at ﬁnal analysis level and corresponding signal simulations from [1] and [22]. JCAP04(2016)022 2.3 Signal and background simulation Solar WIMP signals are simulated using WIMPSim [25], which describes the annihilation of WIMPs inside the Sun. WIMPSim simulates the production, interaction, oscillation and propagation of all three ﬂavours of neutrinos from the core of the Sun to the detector. Muons arising in single or coincident air showers as well as atmospheric neutrinos form the background to this analysis. We did not simulate these contributions, as they can be estimated by scrambling real data at the ﬁnal analysis level (detailed within section 2.7). 2Online data release of ref. [1], available at http://icecube.wisc.edu/science/data/IC79 solarWIMP data release. 2.1 The IceCube detector Completed in December 2010, the IceCube neutrino observatory [23] is a neutrino telescope situated at the South Pole. IceCube is installed in the glacial ice at depths of between 1450 m and 2450 m, instrumenting a total volume of one cubic kilometre. Digital Optical Modules (DOMs) arranged on vertical strings deep in the ice sheet record the Cherenkov light induced by relativistic charged particles, including those created by neutrinos interacting with the ice. The detection of photon yields and arrival times in DOMs allows for the reconstruction of the directions and energies of the secondaries. In its 79-string conﬁguration, 73 strings have a horizontal spacing of 125 m and a vertical spacing of 17 m between DOMs. The six remaining strings are located near the central string of IceCube and feature a reduced vertical spacing between DOMs of 7 m and higher quantum eﬃciency photomultiplier tubes. Along with the seven surrounding regular strings, they form the DeepCore subarray [24]. The horizontal distance between strings in DeepCore is less than 75 m. The higher sensor density in clear ice provides an order of magnitude lower energy threshold of O(10) GeV compared to the main IceCube array. – 2 – Data samples 2.4 Calculation of detector eﬃciencies The eﬀective volume Veﬀ(Eµ) of the detector for muon or anti-muon events produced through charged current interactions diﬀers for each of the three event selections of ref. [1]. Veﬀ(Eµ) for the detection of muons from the Sun is a function of muon energy, averaged over the live-time of the respective event selections. It corresponds to an equivalent volume of 100% detection eﬃciency, and is identical for both muons and anti-muons. We also calculated the eﬀective area Aeﬀ(E) for detection of muon neutrinos as a function of neutrino energy. We use Aeﬀ(E) later to compute ‘bias factors’, which account for selection eﬀects in the analysis (see section 3.6). The eﬀective areas for muon neutrinos and muon anti-neutrinos diﬀer due to the diﬀerences in the (anti-)neutrino cross-sections with hadrons. All eﬀective volumes and areas for the 79-string analysis are available online.2 We specify the total systematic uncertainties related to the detector response at the 1 σ conﬁdence level within each energy bin, in a manner similar to how it was done in Paper I. These uncertainties come from simulation studies, where identiﬁed sources of uncertainty, – 3 – e.g. absolute DOM eﬃciency, photon propagation in ice, or calibration constants, were indi- vidually varied within reasonable ranges of their original values. Similarly, the uncertainties arising from limited simulation statistics are also given for each energy bin of Veﬀ, at the 1 σ conﬁdence level. In the ﬁnal analysis we combine these two errors in quadrature. 2.5 Angular response The point spread function (PSF) describes the uncertainty in the reconstructed arrival direc- tion of muons. Closely following Paper I, the reduced (one-dimensional) PSF for the angular deviation ∆between the true arrival direction of a muon on the sky and its reconstructed direction is 2 P(∆) = ∆ σ2µ exp −∆2 2σ2µ . (2.1) JCAP04(2016)022 (2.1) We extract the parameter σµ, which we refer to as the ‘mean angular error’, directly from the one-dimensional PSF constructed from IceCube signal simulations. As in Paper I, we determine σµ in the same energy bins that were used for calculating the detector eﬃciencies. For simplicity, we neglect the curvature of the PSF on the sky, owing to the fact that for dark matter signals detected with DeepCore, the muon production angle is typically expected to be the dominant source of angular deviation. We therefore restrict our analyses to signal regions of radii φ′ cut around the solar position on the sky so as to minimise the error induced by this approximation (and the fact that we include the entire sky in a data-driven estimation of the background; cf. section 2.7). We determined that φ′ cut = 20◦provides satisfactory signal acceptance and background rejection for the WH sample, and φ′ cut = 40◦is appropriate for the WL and SL datasets. We associate angular uncertainties with real data events on an event-by-event basis, using the paraboloid method [26]. A paraboloid function is ﬁtted to the muon track re- construction likelihood space in the neighbourhood of the best ﬁt. The resulting conﬁdence ellipse on the sky is represented by the two principal axes, which correspond to the standard deviations of the likelihood function in each of two linearly-independent directions. The over- all reconstructed likelihood track uncertainty, σpara (the ‘paraboloid sigma’), is calculated as the mean in quadrature of the uncertainties along the two axes. Good track ﬁts generally result in paraboloids that are narrow along both axes and therefore have small σpara values. 2.6 Energy estimator Paper I used the number of lit DOMs (Nchan) as a suitable energy estimator. This deﬁnition worked well for a detector with a consistent density of optical modules, like the 22-string conﬁguration of IceCube. This paper uses data recorded in the 79-string conﬁguration of IceCube. This conﬁguration includes the DeepCore subarray, which has a higher density of DOMs than the rest of the detector (section 2.1). A simple count of lit DOMs would yield diﬀerent results depending on whether the event crosses, partially crosses, or is contained within DeepCore. In an attempt to address this, we introduce a modiﬁed Nchan value, Nc chan, which corrects for the variation in DOM density across the detector. In this context, the corrected energy proxy Nc chan is Nc chan = NIC chan + fDC × NDC chan , (2.2) (2.2) where NDC chan and NIC chan are the number of lit DOMs in DeepCore (‘standard’ IceCube and ‘high quantum eﬃciency’ DOMs) and the remainder of IceCube, respectively. The factor where NDC chan and NIC chan are the number of lit DOMs in DeepCore (‘standard’ IceCube and ‘high quantum eﬃciency’ DOMs) and the remainder of IceCube, respectively. The factor – 4 – fDC = 0.28 is the the ratio of the number of ‘standard’ IceCube DOMs inside DeepCore to the total number of DOMs in DeepCore, multiplied by an additional correction factor. The ratio accounts for the higher density of DOMs in DeepCore compared to the rest of the detector, and the additional correction factor accounts for the higher quantum eﬃciency of the photomultiplier tubes in the ‘high quantum eﬃciency’ DOMs. We calculated the expected distributions of observed Nc chan values for a series of intervals in muon energy, as we did in neutrino energy in Paper I. Figures 1–3 show these probability distributions for each event selection (WH, WL and SL) and muon energy range. The total interval in muon energy is diﬀerent for each event selection due to the respective event selection criteria that are applied. We use these probability distributions, together with the predicted energy spectrum of the signal from each WIMP model, to calculate the predicted distribution of Nc chan. The ﬁtted functions in ﬁgures 1–3 are only to guide the eye; our signal predictions and likelihood calculations employ the actual distributions. 2.6 Energy estimator The lower plots in ﬁgures 1–3 compare the ﬁtted functions for each event selection, illustrating the ability to diﬀerentiate events between diﬀerent energy intervals. To reach energies as low as the ﬁrst interval in ﬁgures 2 and 3, DeepCore uses an independent, low-threshold, simple majority trigger (SMT), with a 2.5 µs time window, applied to DOMs comprising the DeepCore ﬁducial volume [24]. This trigger requires that three or more DOMs satisfy the so-called hard local coincidence (HLC) condition (as opposed to the threshold of eight or more DOMs, more typically used in IceCube analyses). DOMs meet the HLC condition when two or more DOMs in close proximity to each other (nearest or next-to-nearest neighbours on the same string) register hits within a 1 µs time window. This trigger is 70% eﬃcient for a simulated sample of atmospheric νµ events of 10 GeV neutrino energy [24]. JCAP04(2016)022 More advanced energy reconstruction methods other than Nc chan are available in IceCube that are based on the reconstruction of charged-particle energies and topologies from the observed Cherenkov light yield [27]. Here we use Nc chan for simplicity and robustness. 2.7 Background estimation As in Paper I, the background distributions for each event selection come directly from data. The angular distribution of background events dPBG(φ′)/dφ′ is a function of φ′, the angle be- tween the reconstructed track direction and the Sun. Muons produced in cosmic-ray showers are the dominant contributors to the background. Their angular distribution is observed to be largely independent of azimuth, so we estimated dPBG(φ′)/dφ′ from real data events at the ﬁnal selection level with scrambled azimuths. We used all observed events at the ﬁnal selec- tion level for this exercise. Given the tight upper limit on a signal contribution in the original analysis [1], including the nominal signal region does not bias the background estimate. We calculated the distribution of Nc chan due to background events, dPBG(Nc chan)/dNc chan, and observed no signiﬁcant correlation between the arrival angles of events relative to the Sun and their measured Nc chan values. 2.8 Data format, public code and availability Full event data from the analysis of ref. [1], including angles, Nc chan values and paraboloid sig- mas, can be found at http://icecube.wisc.edu/science/data/IC79 solarWIMP data release. Eﬀective areas and volumes, along with Nc chan and angular responses, can be found at the same location. The nulike code can be downloaded from http://nulike.hepforge.org. The release of nulike coincides with the release of DarkSUSY v5.1.3. This release of DarkSUSY provides optimised – 5 – JCAP04(2016)022 0 20 40 60 80 Probability 0 0.05 0.1 0.15 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 20 40 60 80 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 0 20 40 60 80 0 0.05 0.1 0.15 )/GeV) < 2.0 µ log10(E( ≤ 1.8 0 20 40 60 80 0 0.05 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 0 20 40 60 80 Probability 0 0.02 0.04 0.06 0.08 )/GeV) < 2.4 µ log10(E( ≤ 2.2 0 20 40 60 80 0 0.02 0.04 0.06 )/GeV) < 2.6 µ log10(E( ≤ 2.4 0 20 40 60 80 0 0.02 0.04 0.06 )/GeV) < 2.8 µ log10(E( ≤ 2.6 0 20 40 60 80 0 0.02 0.04 0.06 )/GeV) < 3.0 µ log10(E( ≤ 2.8 chan,c N 0 20 40 60 80 Probability 0 0.02 0.04 )/GeV) < 3.2 µ log10(E( ≤ 3.0 chan,c N 0 20 40 60 80 0 0.02 0.04 )/GeV) < 3.4 µ log10(E( ≤ 3.2 chan,c N 0 20 40 60 80 0 0.01 0.02 0.03 0.04 )/GeV) < 3.6 µ log10(E( ≤ 3.4 chan,c N 0 20 40 60 80 0 0.01 0.02 0.03 0.04 )/GeV) < 4.0 µ log10(E( ≤ 3.8 (a) chan,c N 10 20 30 40 50 60 Probability 0 0.05 0.1 0.15 0.2 )/GeV) < 4.0 µ log10(E( ≤ 3.8 )/GeV) < 3.8 µ log10(E( ≤ 3.6 )/GeV) < 3.6 µ log10(E( ≤ 3.4 )/GeV) < 3.4 µ log10(E( ≤ 3.2 )/GeV) < 3.2 µ log10(E( ≤ 3.0 )/GeV) < 3.0 µ log10(E( ≤ 2.8 )/GeV) < 2.8 µ log10(E( ≤ 2.6 )/GeV) < 2.6 µ log10(E( ≤ 2.4 )/GeV) < 2.4 µ log10(E( ≤ 2.2 )/GeV) < 2.2 µ log10(E( ≤ 2.0 )/GeV) < 2.0 µ log10(E( ≤ 1.8 )/GeV) < 1.8 µ log10(E( ≤ 1.6 )/GeV) < 1.6 µ log10(E( ≤ 1.4 (b) Figure 1. 2.8 Data format, public code and availability Predicted probability distributions of N c chan for the WH event selection, derived from high-statistics simulations used in [1]. Each distribution is deﬁned for muons having energies in a speciﬁc logarithmic energy interval of width 0.2. The ﬁtted functions are to guide the eye only and are not used in our calculations. The lower plot compares the ﬁtted functions, illustrating the ability to diﬀerentiate events between diﬀerent energy intervals. 2.8 Data format, public code and availability 0 20 40 60 80 Probability 0 0.05 0.1 0.15 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 20 40 60 80 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 0 20 40 60 80 0 0.05 0.1 0.15 )/GeV) < 2.0 µ log10(E( ≤ 1.8 0 20 40 60 80 0 0.05 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 0 20 40 60 80 Probability 0 0.05 0.1 0.15 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 20 40 60 80 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 0 20 40 60 80 0 0.05 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 0 20 40 60 80 Probability 0 0.02 0.04 0.06 0.08 )/GeV) < 2.4 µ log10(E( ≤ 2.2 0 20 40 60 80 0 0.02 0.04 0.06 )/GeV) < 2.6 µ log10(E( ≤ 2.4 0 20 40 60 80 0 0.02 0.04 0.06 )/GeV) < 2.8 µ log10(E( ≤ 2.6 0 20 40 60 80 0 0.02 0.04 0.06 )/GeV) < 3.0 µ log10(E( ≤ 2.8 JCAP04(2016)022 chan,c N 0 20 40 60 80 0 0.02 0.04 )/GeV) < 3.4 µ log10(E( ≤ 3.2 chan,c N 0 20 40 60 80 0 0.01 0.02 0.03 0.04 )/GeV) < 3.6 µ log10(E( ≤ 3.4 (a) chan,c N 0 20 40 60 80 Probability 0 0.02 0.04 )/GeV) < 3.2 µ log10(E( ≤ 3.0 chan,c N 0 20 40 60 80 0 0.01 0.02 0.03 0.04 )/GeV) < 4.0 µ log10(E( ≤ 3.8 (a) chan,c N 10 20 30 40 50 60 Probability 0 0.05 0.1 0.15 0.2 )/GeV) < 4.0 µ log10(E( ≤ 3.8 )/GeV) < 3.8 µ log10(E( ≤ 3.6 )/GeV) < 3.6 µ log10(E( ≤ 3.4 )/GeV) < 3.4 µ log10(E( ≤ 3.2 )/GeV) < 3.2 µ log10(E( ≤ 3.0 )/GeV) < 3.0 µ log10(E( ≤ 2.8 )/GeV) < 2.8 µ log10(E( ≤ 2.6 )/GeV) < 2.6 µ log10(E( ≤ 2.4 )/GeV) < 2.4 µ log10(E( ≤ 2.2 )/GeV) < 2.2 µ log10(E( ≤ 2.0 )/GeV) < 2.0 µ log10(E( ≤ 1.8 )/GeV) < 1.8 µ log10(E( ≤ 1.6 )/GeV) < 1.6 µ log10(E( ≤ 1.4 (b) (b) Figure 1. Predicted probability distributions of N c chan for the WH event selection, derived from high-statistics simulations used in [1]. 2.8 Data format, public code and availability Each distribution is deﬁned for muons having energies in a speciﬁc logarithmic energy interval of width 0.2. The ﬁtted functions are to guide the eye only and are not used in our calculations. The lower plot compares the ﬁtted functions, illustrating the ability to diﬀerentiate events between diﬀerent energy intervals. – 6 – JCAP04(2016)022 0 5 10 15 20 Probability 0 0.1 0.2 0.3 )/GeV) < 0.8 µ log10(E( ≤ 0.6 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.0 µ log10(E( ≤ 0.8 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.2 µ log10(E( ≤ 1.0 0 5 10 15 20 0 0.1 0.2 )/GeV) < 1.4 µ log10(E( ≤ 1.2 0 5 10 15 20 Probability 0 0.1 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 5 10 15 20 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 0 5 10 15 20 0 0.05 0.1 0.15 )/GeV) < 2.0 µ log10(E( ≤ 1.8 0 5 10 15 20 0 0.05 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 chan,c N 0 5 10 15 20 Probability 0 0.05 0.1 )/GeV) < 2.4 µ log10(E( ≤ 2.2 chan,c N 0 5 10 15 20 0 0.05 0.1 )/GeV) < 2.6 µ log10(E( ≤ 2.4 chan,c N 0 5 10 15 20 0 0.05 0.1 )/GeV) < 2.8 µ log10(E( ≤ 2.6 chan,c N 0 5 10 15 20 0 0.05 0.1 )/GeV) µ log10(E( ≤ 2.8 (a) chan,c N 5 10 15 20 Probability 0 0.1 0.2 0.3 0.4 )/GeV) µ log10(E( ≤ 2.8 )/GeV) < 2.8 µ log10(E( ≤ 2.6 )/GeV) < 2.6 µ log10(E( ≤ 2.4 )/GeV) < 2.4 µ log10(E( ≤ 2.2 )/GeV) < 2.2 µ log10(E( ≤ 2.0 )/GeV) < 2.0 µ log10(E( ≤ 1.8 )/GeV) < 1.8 µ log10(E( ≤ 1.6 )/GeV) < 1.6 µ log10(E( ≤ 1.4 )/GeV) < 1.4 µ log10(E( ≤ 1.2 )/GeV) < 1.2 µ log10(E( ≤ 1.0 )/GeV) < 1.0 µ log10(E( ≤ 0.8 )/GeV) < 0.8 µ log10(E( ≤ 0.6 (b) Figure 2. As per ﬁgure 1, but for the WL event selection. 2.8 Data format, public code and availability 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.0 µ log10(E( ≤ 0.8 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.2 µ log10(E( ≤ 1.0 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.0 µ log10(E( ≤ 0.8 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.2 µ log10(E( ≤ 1.0 0 5 10 15 20 Probability 0 0.1 0.2 0.3 )/GeV) < 0.8 µ log10(E( ≤ 0.6 0 5 10 15 20 0 0.1 0.2 )/GeV) < 1.4 µ log10(E( ≤ 1.2 0 5 10 15 20 Probability 0 0.1 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 5 10 15 20 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 0 5 10 15 20 0 0.05 0.1 0.15 )/GeV) < 2.0 µ log10(E( ≤ 1.8 0 5 10 15 20 0 0.05 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 JCAP04(2016)022 chan,c N 0 5 10 15 20 Probability 0 0.05 0.1 )/GeV) < 2.4 µ log10(E( ≤ 2.2 chan,c N 0 5 10 15 20 0 0.05 0.1 )/GeV) < 2.6 µ log10(E( ≤ 2.4 chan,c N 0 5 10 15 20 0 0.05 0.1 )/GeV) < 2.8 µ log10(E( ≤ 2.6 (a) chan,c N 0 5 10 15 20 0 0.05 0.1 )/GeV) µ log10(E( ≤ 2.8 (a) chan,c N 5 10 15 20 Probability 0 0.1 0.2 0.3 0.4 )/GeV) µ log10(E( ≤ 2.8 )/GeV) < 2.8 µ log10(E( ≤ 2.6 )/GeV) < 2.6 µ log10(E( ≤ 2.4 )/GeV) < 2.4 µ log10(E( ≤ 2.2 )/GeV) < 2.2 µ log10(E( ≤ 2.0 )/GeV) < 2.0 µ log10(E( ≤ 1.8 )/GeV) < 1.8 µ log10(E( ≤ 1.6 )/GeV) < 1.6 µ log10(E( ≤ 1.4 )/GeV) < 1.4 µ log10(E( ≤ 1.2 )/GeV) < 1.2 µ log10(E( ≤ 1.0 )/GeV) < 1.0 µ log10(E( ≤ 0.8 )/GeV) < 0.8 µ log10(E( ≤ 0.6 (b) (b) Figure 2. As per ﬁgure 1, but for the WL event selection. 2.8 Data format, public code and availability – 7 – 0 5 10 15 20 Probability 0 0.1 0.2 0.3 )/GeV) < 0.8 µ log10(E( ≤ 0.6 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.0 µ log10(E( ≤ 0.8 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.2 µ log10(E( ≤ 1.0 0 5 10 15 20 Probability 0 0.05 0.1 0.15 0.2 0.25 )/GeV) < 1.4 µ log10(E( ≤ 1.2 0 5 10 15 20 0 0.05 0.1 0.15 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 5 10 15 20 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 chan c N 0 5 10 15 20 Probability 0 0.02 0.04 0.06 0.08 0.1 0.12 )/GeV) < 2.0 µ log10(E( ≤ 1.8 chan c N 0 5 10 15 20 0 0.02 0.04 0.06 0.08 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 chan c N 0 5 10 15 20 0 0.02 0.04 0.06 0.08 )/GeV) µ log10(E( ≤ 2.2 (a) chan c N 0 5 10 15 20 Probability 0.1 0.2 0.3 )/GeV) µ log10(E( ≤ 2.2 )/GeV) < 2.2 µ log10(E( ≤ 2.0 )/GeV) < 2.0 µ log10(E( ≤ 1.8 )/GeV) < 1.8 µ log10(E( ≤ 1.6 )/GeV) < 1.6 µ log10(E( ≤ 1.4 )/GeV) < 1.4 µ log10(E( ≤ 1.2 )/GeV) < 1.2 µ log10(E( ≤ 1.0 )/GeV) < 1.0 µ log10(E( ≤ 0.8 )/GeV) < 0.8 µ log10(E( ≤ 0.6 (b) Figure 3. As per ﬁgure 1, but for the SL event selection. 3.1 General form The primary improvement in the likelihood treatment here compared to Paper I [7] is that we allow for diﬀerences between the arrival directions of neutrinos (φ) and the muons they produce (φµ). At neutrino energies above O(100 GeV), to a good approximation one can neglect the diﬀerence between φ and φµ. This was the case for all data and calculations considered in Paper I. With the DeepCore inﬁll array however, the actual 79- and 86-string IceCube conﬁgurations are sensitive to neutrino energies even below 10 GeV. For example, for a neutrino of energy 10 GeV producing a muon of 4 GeV, φ −φµ can be as large as 30 degrees, and must therefore be explicitly included in all calculations. JCAP04(2016)022 The distribution of muon production angles introduces an explicit energy dependence to the detector PSF. This improves on our earlier approximation that the detector response factorises into separate functions of angle and energy (eq. (3.6) in Paper I). In this paper we therefore work with the general form of the unbinned likelihood, Lunbin ≡Lnum(ntot\|θtot) ntot Y i=1 Z π 0 Z ∞ 0 Q(Nc i , φ′ i\|E, φ) d2P dE dφ(E, φ,⃗ξ⃗ξ⃗ξ) dE dφ . (3.1) (3.1) The vector ⃗ξ⃗ξ⃗ξ refers to the parameters of a given BSM model. Nc i and φ′ i are the actual observed event-level data for the ith event of ntot total events. Nc i in this analysis is the generalised Nchan, whereas φ′ i is the angle between the reconstructed muon track and the direction of the Sun. As in Paper I, Q(Nc i , φ′ i\|E, φ) is the probability density (in eﬀective units of inverse angle and Nc chan) for observing Nc i and φ′ i for the ith event when the true values of the incoming neutrino energy and angle relative to the Sun are E and φ, respectively. values of the incoming neutrino energy and angle relative to the Sun are E and φ, respectively. The prefactor Lnum is the number likelihood for observing ntot events given a prediction θtot, marginalised over the systematic error on the predicted number of events. This is The prefactor Lnum is the number likelihood for observing ntot events given a prediction θtot, marginalised over the systematic error on the predicted number of events. This is Lnum(ntot\|θtot) = 1 √ 2πσϵ Z ∞ 0 (θBG + ϵθS)ntote−(θBG+ϵθS) ntot! 2.8 Data format, public code and availability 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.0 µ log10(E( ≤ 0.8 0 5 10 15 20 Probability 0 0.1 0.2 0.3 )/GeV) < 0.8 µ log10(E( ≤ 0.6 0 5 10 15 20 0 0.1 0.2 0.3 )/GeV) < 1.2 µ log10(E( ≤ 1.0 0 5 10 15 20 Probability 0 0.05 0.1 0.15 0.2 0.25 )/GeV) < 1.4 µ log10(E( ≤ 1.2 0 5 10 15 20 0 0.05 0.1 0.15 0.2 )/GeV) < 1.6 µ log10(E( ≤ 1.4 0 5 10 15 20 0 0.05 0.1 0.15 )/GeV) < 1.8 µ log10(E( ≤ 1.6 JCAP04(2016)022 chan c N 0 5 10 15 20 0 0.02 0.04 0.06 0.08 0.1 )/GeV) < 2.2 µ log10(E( ≤ 2.0 (a) chan c N 0 5 10 15 20 0 0.02 0.04 0.06 0.08 )/GeV) µ log10(E( ≤ 2.2 chan c N 0 5 10 15 20 Probability 0 0.02 0.04 0.06 0.08 0.1 0.12 )/GeV) < 2.0 µ log10(E( ≤ 1.8 (a) chan c N 0 5 10 15 20 Probability 0.1 0.2 0.3 )/GeV) µ log10(E( ≤ 2.2 )/GeV) < 2.2 µ log10(E( ≤ 2.0 )/GeV) < 2.0 µ log10(E( ≤ 1.8 )/GeV) < 1.8 µ log10(E( ≤ 1.6 )/GeV) < 1.6 µ log10(E( ≤ 1.4 )/GeV) < 1.4 µ log10(E( ≤ 1.2 )/GeV) < 1.2 µ log10(E( ≤ 1.0 )/GeV) < 1.0 µ log10(E( ≤ 0.8 )/GeV) < 0.8 µ log10(E( ≤ 0.6 (b) (b) Figure 3. As per ﬁgure 1, but for the SL event selection. – 8 – interpolation routines for WIMPSim [25] outputs contained in DarkSUSY, and ensures that they are fully compatible with the parallel likelihood routines in nulike (i.e. the routines one would use together with nulike are threadsafe in the latest DarkSUSY release). 3.1 General form 1 ϵ exp " −1 2 ln ϵ σϵ 2# dϵ , (3.2) (3.2) where θS is the predicted number of signal events, θBG is the predicted number of background events, θtot = θS + θBG, ϵ is the rescaling variable assumed to have a log-normal distribution, and σϵ is the fractional systematic error on the signal prediction (which sets the width of the distribution of ϵ). The width σϵ is the sum in quadrature of a theoretical error τ and the fractional uncertainty on the detector response. This treatment requires the selection of a single indicative systematic error on the eﬀective volume, which is then applied identically at all muon energies. When computing results, to be conservative we chose the largest systematic error on the eﬀective volume over the entire range of detectable muon energies. For the theoretical error τ we adopted a minimum of 5% for WIMP masses mχ ≤100 GeV to account for neglected higher order corrections and round-oﬀerrors, increasing to 50% at mχ = 10 TeV as mχ = 10 TeV as τ = 0.05 mχ 100 GeV 1/2 . (3.3) (3.3) – 9 – This sliding scale is designed to encapsulate the increasing error with WIMP mass of predicted spectra from DarkSUSY, due to internal tables in which it interpolates results from WIMPSim. Paper I and refs. [16, 28] give further details and background on the number likelihood. , p aper I and refs. [16, 28] give further details and background on the number likelihood. The expected distribution of incident neutrino energies (E) and angles (φ) is given by d2P/dE dφ(E, φ,⃗ξ⃗ξ⃗ξ), which is a prediction of the model parameters ⃗ξ⃗ξ⃗ξ. This separates into a weighted sum of the signal (S) and background (BG) contributions, so that eq. (3.1) can be expressed as Lunbin ≡Lnum(ntot\|θtot) ntot Y i=1 (fSLS,i + fBGLBG,i) , (3.4) (3.4) where fS ≡θS/θtot and fBG ≡θBG/θtot are the fractions of the total expected events from signal and background, respectively, and JCAP04(2016)022 LX,i(Nc i , φ′ i\|⃗ξ⃗ξ⃗ξ) ≡ Z π 0 Z ∞ 0 Q(Nc i , φ′ i\|E, φ) d2PX dE dφ(E, φ,⃗ξ⃗ξ⃗ξ) dE dφ , (3.5) (3.5) gives the signal (X = S) and background likelihoods (X = BG). gives the signal (X = S) and background likelihoods (X = BG). gives the signal (X = S) and background likelihoods (X = BG). 3.2 Background likelihood The calculation of the background likelihood component follows the treatment in Paper I closely: the integral in eq. (3.5) for X = BG is the actual observed background, which is independent of the model parameters ⃗ξ⃗ξ⃗ξ. Within the zenith angle range considered in this analysis, to a very good approximation, the background spectrum and angular distributions are not correlated. LBG,i can then be written as LBG,i(Nc i , φ′ i) = dPBG dNc i (Nc i )dPBG dφ′ i (φ′ i) , (3.6) (3.6) where dPBG/dNc i and dPBG/dφ′ i are the observed Nc chan and angular distributions of the background, respectively (section 2.7). The expected number of background events θBG used to calculate the background fraction fBG refers to the events contained in the angular cut φ′ cut around the solar position. where dPBG/dNc i and dPBG/dφ′ i are the observed Nc chan and angular distributions of the background, respectively (section 2.7). The expected number of background events θBG used to calculate the background fraction fBG refers to the events contained in the angular cut φ′ cut around the solar position. 3.3 Signal likelihood (3 7) JCAP04(2016)022 # (3.7) Here φµ is the angle of the produced muon relative to the Sun, Eµ is its energy, and barred quantities are the equivalent measures for anti-particles. The angular component of the signal prediction is a delta function at the solar position d2Φν dE dφ(E, φ,⃗ξ⃗ξ⃗ξ) = dΦν dE (E,⃗ξ⃗ξ⃗ξ)δ(φ) , (3.8) (3.8) so the integral of eq. (3.7) over φ (required by eq. (3.5) in order to obtain the signal likelihood) can be done analytically. We then ﬁnd so the integral of eq. (3.7) over φ (required by eq. (3.5) in order to obtain the signal likelihood) can be done analytically. We then ﬁnd LS,i(Nc i ,φ′ i\|⃗ξ⃗ξ⃗ξ) = texp θS X ν,¯ν Z ∞ 0 dΦν dE (E,⃗ξ⃗ξ⃗ξ)fb ν (E) Z ∞ 0 Z π 0 Qµ(Nc i ,φ′ i\|Eµ,φµ)d2Σν→µ dEµdφµ (Eµ,φµ\|E,0)dφµdEµdE, (3 9) texp θS X ν,¯ν Z ∞ 0 dΦν dE (E,⃗ξ⃗ξ⃗ξ)fb ν (E) Z ∞ 0 Z π 0 Qµ(Nc i ,φ′ i\|Eµ,φµ)d2Σν→µ dEµdφµ (Eµ,φµ\|E,0)dφµdEµdE, (3 9 (3.9) where the sum indicates that the corresponding antiparticle expression must also be included. With φ = 0, the true muon arrival angle relative to the Sun, φµ, becomes identical to the microscopic muon production angle in the frame where the target nucleus is at rest. The value of this angle depends on the incoming neutrino energy and the outgoing muon energy, as well as the momentum carried by the parton within the nucleon with which the neutrino interacts. It can be written as cos φµ(x, y, E) = E2 µ −m2 µ −1/2 Eµ −mNxy −m2 µ/2E , (3.10) (3.10) where mN refers to the mass of the nucleon involved. The Bj¨orken scaling variable x indicates the fraction of the nucleonic momentum carried by the parton involved in the interaction. By deﬁnition, x varies between 0 and 1, as does the other Bj¨orken variable y = 1 −Eµ/E. Together, x and y provide a convenient and well-bounded way to express the dependence of the neutrino interaction cross-sections on the outgoing muon energy and angle. 3.3 Signal likelihood In order to take into account the distribution of production angles in calculating the signal likelihood, the integral in eq. (3.5) for X = S should be expressed in terms of the kinematics of the produced muons. In eq. (3.5) this integrand is the product of the predicted arrival probability of a neutrino of a given energy and arrival angle, and the detector response to it. We express this as the product of the predicted diﬀerential ﬂux of incoming neutrinos (d2Φν/dE dφ), the exposure time of the observation (texp), the eﬀective diﬀerential cross- section for neutrino conversion into muons in the ice (d2Σν→µ/dEµ dφµ), and the response of the detector to muon-conversion events (Qµ). We then integrate over the distribution of muon energies and angles that might be created in the interaction, so as to recover a pure function of the neutrino properties (as the theoretical predictions of diﬀerent dark matter models ⃗ξ⃗ξ⃗ξ are given at neutrino level). We divide by the expected number of signal events θS inside the angular cut cone, in order to normalise the integral of the resulting probability distribution to unity. We also multiply by a bias factor fb(E), which is an analysis-dependent – 10 – function of the neutrino energy. θS and fb(E) are discussed in detail in section 3.5. Finally, we add the contributions of both incoming neutrinos and antineutrinos, giving: function of the neutrino energy. θS and fb(E) are discussed in detail in section 3.5. Finally, we add the contributions of both incoming neutrinos and antineutrinos, giving: Q(Nc i , φ′ i\|E, φ) d2PS dE dφ(E, φ,⃗ξ⃗ξ⃗ξ) = texp θS " d2Φν dE dφ(E, φ,⃗ξ⃗ξ⃗ξ)fb ν (E) Z ∞ 0 Z φ+π φ Qµ(Nc i , φ′ i\|Eµ, φµ) d2Σν→µ dEµ dφµ (Eµ, φµ\|E, φ) dφµ dEµ + d2Φ¯ν dE dφ(E, φ,⃗ξ⃗ξ⃗ξ)fb ¯ν (E) Z ∞ 0 Z φ+π φ Q¯µ(Nc i , φ′ i\|E¯µ, φ¯µ) d2Σ¯ν→¯µ dE¯µ dφ¯µ (E¯µ, φ¯µ\|E, φ) dφ¯µ dE¯µ # . (3 7) exp θS " d2Φν dE dφ(E, φ,⃗ξ⃗ξ⃗ξ)fb ν (E) Z ∞ 0 Z φ+π φ Qµ(Nc i , φ′ i\|Eµ, φµ) d2Σν→µ dEµ dφµ (Eµ, φµ\|E, φ) dφµ dEµ + d2Φ¯ν dE dφ(E, φ,⃗ξ⃗ξ⃗ξ)fb ¯ν (E) Z ∞ 0 Z φ+π φ Q¯µ(Nc i , φ′ i\|E¯µ, φ¯µ) d2Σ¯ν→¯µ dE¯µ dφ¯µ (E¯µ, φ¯µ\|E, φ) dφ¯µ dE¯µ # . 3.3 Signal likelihood We therefore trade Eµ and φµ for x and y, so that LS,i(Nc i , φ′ i\|⃗ξ⃗ξ⃗ξ) becomes texp θS X ν,¯ν Z ∞ 0 dΦν dE (E,⃗ξ⃗ξ⃗ξ)fb ν (E) ZZ 1 0 Qµ Nc i , φ′ i\|Eµ, φµ d2Σν→µ dx dy (x, y\|E) dx dy dE , (3.11) (3.11) remembering that Eµ = Eµ(y, E) and φµ = φµ(x, y, E). For each observed event inside our analysis cone, we precompute the inner double integral of eq. (3.11) for a set of 50 logarithmically-spaced neutrino energies per decade over the range 0.5 ≤log10(E/GeV) ≤ – 11 – 4.0. To obtain the contribution of the predicted signal to the total likelihood for that event, we re-weight these ‘partial likelihoods’ according to the predicted neutrino spectrum for each model ⃗ξ⃗ξ⃗ξ, as well as the bias factor fb. To allow for a fast and straightforward application to any theoretical neutrino spectrum, we provide the partial likelihoods for the 79-string IceCube analysis in nulike, precomputed, along with routines for computing the bias factors fb. We also provide the underlying event data online2 and a utility within nulike that can precompute and save the partial likelihoods from any other neutrino telescope, provided the data are in the same format. The eﬀective diﬀerential conversion cross-section is given by d2Σν→µ dx dy (x, y\|E) = Veﬀ(Eµ) X N=p,n nN d2σν→µ,N dx dy (x, y\|E) , (3.12) (3.12) JCAP04(2016)022 where it should again be understood that Eµ = Eµ(y, E). The replacement {ν, µ} →{¯ν, ¯µ} provides the corresponding expression for d2Σ¯ν→¯µ/dx dy. This is the product of the number density nN of nucleon species N (proton or neutron) in the detector, the eﬀective volume Veﬀ(Eµ) of the detector for muon or anti-muon conversion events, and d2σν→µ,N/dx dy, the microscopic diﬀerential cross-section for muon production by charged-current interactions. Veﬀis the same for both muons and anti-muons. In contrast, the diﬀerential cross-sections diﬀer for particles and antiparticles. These are known from the theory of weak interactions, up to a dependence on the parton distributions for x. We obtain these from nusigma [29], which by default relies on the CTEQ6-DIS parton distribution functions [30]. Users of nulike who prefer other parton distributions can simply switch those employed by nusigma and recompute the partial likelihoods. 3.5 Predicted event rate The total predicted number of signal events θS follows similarly to eq. (3.11) as the sum of the predicted number of neutrino-initiated signal events The total predicted number of signal events θS follows similarly to eq. (3.11) as the sum of the predicted number of neutrino-initiated signal events θS,ν(⃗ξ⃗ξ⃗ξ) = texp Z ∞ 0 dΦν dE (E,⃗ξ⃗ξ⃗ξ)fb ν (E) ZZ 1 0 L(φµ, Eµ, φ′ cut)d2Σν→µ dx dy (x, y\|E) dx dy dE , (3.15) (3.15) and the corresponding quantity θS,¯ν for anti-neutrinos. Again, we remind the reader that φµ and Eµ are functions of x, y and E. The only diﬀerence here with respect to what one would naively read oﬀeq. (3.11) is the factor L(φµ, Eµ, φ′ cut), a dimensionless, energy-dependent angular loss factor that is independent of the muon charge. L corrects for neutrinos that originate from the direction of the Sun but produce muons that are ultimately reconstructed as arriving from outside the analysis cut cone (φ′ i > φ′ cut). Similarly to Paper I, we use the mean angular error of IceCube (σµ; cf. section 2.5) to calculate L, integrating the PSF over the analysis cut cone to give JCAP04(2016)022 L(φµ, Eµ, φ′ cut) = Z φ′ cut 0 φ′ σ2µ exp " −φ′2 + φµ2 2σ2µ # I0 φµφ′ σ2µ dφ′ ≡P1 φµ σµ , φ′ cut σµ . (3.16) (3.16) This is known as the Marcum P-function or Complementary Marcum Q-function, which we evaluate with the code of ref. [31]. There are two crucial diﬀerences here as compared to Paper I. The ﬁrst is that L is a muon-level correction factor, expressed in terms of the muon energy and the width of the muon-level angular uncertainty σµ(Eµ), not the corresponding neutrino quantities. The other is that because of the non-zero muon production angle, the oﬀ-centre PSF (eq. (3.14)) must be used instead of the central distribution (eq. (2.1)). The mean angular error is the correct PSF width to use in eq. (3.16), because we are interested in determining a priori what fraction of incoming neutrinos with a given energy should be absent from the ﬁnal set of observed events, due to the chosen angular cut. This is in contrast to the case of the contribution to the partial likelihood coming from the detector response (eq. 3.4 Detector response Based on the observation that the angular and spectral (Nc chan) distributions of detected events are essentially uncorrelated across the sky (section 2.7), we assume that the detector response to events producing muons factorises into the product Qµ(Nc i , φ′ i\|Eµ, φµ) = Edisp(Nc i \|Eµ)PSF(φ′ i\|φµ, Eµ) . (3.13) (3.13) Here Edisp(Nc i \|Eµ) is the energy dispersion of the detector and PSF(φ′ i\|φµ, Eµ) is its point spread function, assuming these to be identical for muons and antimuons. The energy dis- persion is the Nc chan response to events that produce muons of a given energy (in contrast to the neutrino Nchan response that we employed in Paper I). We obtained this from IceCube detector Monte Carlo simulations (section 2.6). Here Edisp(Nc i \|Eµ) is the energy dispersion of the detector and PSF(φ′ i\|φµ, Eµ) is its point spread function, assuming these to be identical for muons and antimuons. The energy dis- persion is the Nc chan response to events that produce muons of a given energy (in contrast to the neutrino Nchan response that we employed in Paper I). We obtained this from IceCube detector Monte Carlo simulations (section 2.6). The uncertainty in the muon reconstruction direction is given on a per-event basis by the IceCube paraboloid sigma σpara,i for the ith event (section 2.5), which accounts for the dependence of the PSF on the incoming muon energy. To obtain the PSF in terms of φ′ i and φµ, we shift from the coordinate system centred on the true muon arrival direction (i.e. ∆= 0 in eq. (2.1)) to the one with the Sun at the origin (φ′ = 0), integrating over all azimuths to obtain PSF(φ′ i\|φµ, Eµ) = φ′ i σ 2 para,i exp " −φ′ i 2 + φµ2 2σ 2 para,i # I0 φµφ′ i σ 2 para,i ! , (3.14) (3.14) where I0 is the lowest-order modiﬁed Bessel function of the ﬁrst kind.3 3The deﬁnition of the PSF here diﬀers from Paper I, as in the current paper we allow for diﬀerences between the neutrino and muon angles, perform the co-ordinate shift and normalise over the whole sky. Previously, – 12 – – 12 – 3.5 Predicted event rate (3.14)), where we are interested in the probability that a given event originated from the Sun, where the event-level paraboloid σpara,i should be preferred. we normalised over the analysis cut cone instead of the full sky, and normalised with respect to the allowed ranges of the true direction and its deviation from the reconstructed one, rather than by simply considering the permitted values of the reconstructed angle like we do here. This marginally degraded the sensitivity of the previous analysis, although the eﬀect was negligible in comparison to the experimental uncertainty. 3.6 Bias factor calculation The inner double integral in eq. (3.15) gives the unbiased neutrino eﬀective area for this analysis. It diﬀers from the eﬀective area derived in the standard 79-string analysis [1] in two important ways. First, it includes the factor L, to account for the angular loss due to our analysis cut cone around the solar position. Second, it implicitly assumes that all muons of a given energy are equally likely to pass the original analysis cuts used in the 79-string analysis. In reality, low-energy muons created by high-energy neutrinos are, for example, far more likely to appear in the ﬁnal event sample than muons of the same energy created by low-energy neutrinos. This is due to the additional light deposited in the detector from the hadronic recoil in the case of a higher-energy neutrino, and the analysis cuts placed on quantities such as the absolute number of activated DOMs. we normalised over the analysis cut cone instead of the full sky, and normalised with respect to the allowed ranges of the true direction and its deviation from the reconstructed one, rather than by simply considering the permitted values of the reconstructed angle like we do here. This marginally degraded the sensitivity of the previous analysis, although the eﬀect was negligible in comparison to the experimental uncertainty. – 13 – This departure from a perfect mapping between the properties of a muon and its prob- ability of ending up in the ﬁnal event sample constitutes a bias that depends on the neutrino energy. This is precisely the reason for the bias factor fb(E) in the preceding expressions, which accounts for the departure of the the event sample from the minimum bias expectation. To quantify this eﬀect, we take the ratio fb(E) = Aeﬀ(E) ZZ 1 0 d2Σν→µ dx dy (x, y\|E) dx dy −1 , (3.17) (3.17) of the original 79-string eﬀective area Aeﬀ(E) to the unbiased eﬀective area calculated without the angular correction L. The ﬁnal eﬀective area in this paper is the product of the bias factor fb and the unbiased eﬀective area with the angular correction L. In this way, our analysis is fully consistent with the original 79-string eﬀective area by construction, and accounts for both the bias and the angular cut cone at the same time. 3.6 Bias factor calculation JCAP04(2016)022 To facilitate the use of other neutrino spectra, we provide unbiased eﬀective areas pre- computed in nulike for the three 79-string IceCube event selections, both with and without the angular correction L. We also provide the routines necessary to repeat the computations for any other dataset. In ﬁnal likelihood mode, the user can choose to have nulike work with user-supplied bias factors, or use the unbiased eﬀective areas to automatically determine the bias factors. 4 Improved limits on WIMP dark matter At high masses, the combined limit in ﬁgure 4 essentially tracks the exclusion curve of the WH sample, which is orders of magnitude more sensitive than the WL and SL samples – 14 – 101 102 103 104 Dark matter mass mχ (GeV) 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 IC79 τ +τ − SL WL WH full L SL, counting WL, counting WH, counting all, counting Figure 4. Limits on dark matter annihilation in the Sun using an analysis that takes into account neutrino energy information. We show limits separately for the three diﬀerent IC79 event samples SL (summer low) WL (winter low) and WH (winter high) and their combination. The diﬀerence between dashed and solid lines indicates the improvement gained by moving from a simple counts- based number likelihood to a full unbinned one, incorporating the number of events, their arrival directions and energies. The full limit is weaker than the WL sample taken alone at low masses, because the SL sample exhibits a weak excess (< 2σ local signiﬁcance) of events above background expectation, not borne out in the WL sample. Here we have assumed an annihilation cross-section of ⟨σv⟩0 = 3 × 10−26 cm3 s−1 entirely into τ +τ −ﬁnal states. 101 102 103 104 Dark matter mass mχ (GeV) 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 IC79 τ +τ − SL WL WH full L SL, counting WL, counting WH, counting all, counting JCAP04(2016)022 Dark matter mass mχ (GeV) Figure 4. Limits on dark matter annihilation in the Sun using an analysis that takes into account neutrino energy information. We show limits separately for the three diﬀerent IC79 event samples SL (summer low) WL (winter low) and WH (winter high) and their combination. The diﬀerence between dashed and solid lines indicates the improvement gained by moving from a simple counts- based number likelihood to a full unbinned one, incorporating the number of events, their arrival directions and energies. The full limit is weaker than the WL sample taken alone at low masses, because the SL sample exhibits a weak excess (< 2σ local signiﬁcance) of events above background expectation, not borne out in the WL sample. Here we have assumed an annihilation cross-section of ⟨σv⟩0 = 3 × 10−26 cm3 s−1 entirely into τ +τ −ﬁnal states. 4 Improved limits on WIMP dark matter Figures 4–7 show the 90% conﬁdence level (CL) limits on simple eﬀective WIMP DM models computed using IC79 data (section 2) and the nulike 1.0.0 implementation of the likelihood described in section 3. We use the ∆ln L relative to the background-only prediction as the test statistic, summed over the three event selections, conditioning on all parameters except the cross-section to leave only a single degree of freedom. The distribution of this test statistic is very close to χ2, as shown in previous analyses by explicit Monte Carlo [1, 22]; this allows CLs to be determined by standard ∆χ2 methods. For all limits in this section, we assume that DM annihilates exclusively to some speciﬁc ﬁnal state, with a canonical thermal annihilation cross-section ⟨σv⟩0 = 3 × 10−26 cm3 s−1. For all but the highest WIMP masses and lowest scattering cross-sections, these models have reached equilibrium between capture and annihilation in the Sun. We do not assume equilibrium in our calculations however, as is often done. We use DarkSUSY 5.1.3 to compute the predicted neutrino spectrum at the detector for each model, and to solve for the present- day DM population in the Sun. We adopt the standard halo model and default nuclear matrix elements as implemented in DarkSUSY; see discussions in refs. [17, 34]. Figure 4 presents the limits on the spin-dependent WIMP-proton cross-section imposed by the three diﬀerent IC79 event samples: WH, WL and SL individually, and in combination. As an example, here we show limits corresponding to annihilation solely to τ +τ −ﬁnal states. As expected [1, 22], the SL and WL samples dominate the sensitivity at low WIMP masses. For comparison, we also show limits based on the number likelihood (eq. (3.2)) alone, ne- glecting all event-level information. For the cut cone that we use (40 degrees for WL and SL, 20 degrees for WH), considering the arrival directions and energies of neutrino events provides up to a factor of 20 improvement in the resulting limits. 4 Improved limits on WIMP dark matter in this region of parameter space. At masses below 100 GeV however, where SL and WL both play signiﬁcant roles, the combined limit is slightly weaker than the limit obtained by considering the WL sample alone. This is because the SL sample exhibits a weak excess above the background expectation inside the analysis cut cone that is not replicated in the WL sample: 819 observed events as compared to 770 predicted in the analysis cone from background alone. In ﬁgure 5 we compare these new limits to the previous 79-string IceCube constraints on hard and soft annihilation channels. To allow a reasonable comparison, here we show limits for b¯b, W +W −and τ +τ −ﬁnal states, matching what was used in the previous analysis (‘soft channel’ = b¯b, ‘hard channel’ = W +W −for mχ > mW and τ +τ −for mχ < mW ). The previous analysis used the same data as we use here, except that it did not include event – 15 – 101 102 103 104 Dark matter mass mχ (GeV) 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 10−33 10−32 10−31 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 IC79 b¯b, counting b¯b, full L W +W −, counting W +W −, full L τ +τ −, counting τ +τ −, full L PRL soft PRL hard Figure 5. Limits on the spin-dependent WIMP-proton cross-section from IC79 using the improved likelihood, for the canonical soft (b¯b) and hard (W +W −and τ +τ −) annihilation channels often seen in SUSY models. Here we compare to the limits from the original IC79 analysis (‘PRL’; [1]); note that the previous ‘hard’ channel limit is W +W −above the W mass, but τ +τ −below it. The addition of energy information provides an improvement of up to a factor of 4 at high WIMP masses over the previous analysis, whereas the limits are in excellent agreement for low WIMP masses. Here we have assumed an annihilation cross-section of ⟨σv⟩0 = 3 × 10−26 cm3 s−1. 4 Improved limits on WIMP dark matter 101 102 103 104 Dark matter mass mχ (GeV) 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 10−33 10−32 10−31 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 IC79 b¯b, counting b¯b, full L W +W −, counting W +W −, full L τ +τ −, counting τ +τ −, full L PRL soft PRL hard JCAP04(2016)022 102 103 Dark matter mass mχ (GeV) Figure 5. Limits on the spin-dependent WIMP-proton cross-section from IC79 using the improved likelihood, for the canonical soft (b¯b) and hard (W +W −and τ +τ −) annihilation channels often seen in SUSY models. Here we compare to the limits from the original IC79 analysis (‘PRL’; [1]); note that the previous ‘hard’ channel limit is W +W −above the W mass, but τ +τ −below it. The addition of energy information provides an improvement of up to a factor of 4 at high WIMP masses over the previous analysis, whereas the limits are in excellent agreement for low WIMP masses. Here we have assumed an annihilation cross-section of ⟨σv⟩0 = 3 × 10−26 cm3 s−1. energy information in the likelihood function. At low masses, the analysis agrees with the previous one, indicating that the energy information adds little information. Including the event-level energy information has the most impact at high WIMP mass, making use of the relatively good energy resolution of IceCube at high muon energies. The limits in ﬁgure 5 are up to a factor of 4 stronger than the previous analysis at multi-TeV masses. The latest update of WIMPSim ﬁxes an issue with propagation of neutrinos in the Sun that aﬀected the version used to derive the original IC79 limits [1]. This resulted in conservative limits for WIMP masses above ∼500 GeV, ranging from a factor of 1.05 at 500 GeV to 1.2 at 1 TeV and up to 1.5 at 5 TeV for the W +W −and τ +τ −ﬁnal states. Improvements beyond those factors are due to the improved analysis method in this paper. Figure 6 compares these limits to other searches for spin-dependent DM-proton scatter- ing, both from the Sun and direct detection experiments. 4 Improved limits on WIMP dark matter The 79-string IceCube data provide the strongest limits of any search for all masses above ∼100–200 GeV (the exact value de- – 16 – 101 102 103 104 Dark matter mass mχ (GeV) 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 10−33 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 Super-K b¯b Super-K W +W − Super-K τ +τ − PICO-2L (2015) IceCube b¯b IceCube W +W − IceCube τ +τ − PICO-60 (2015) Figure 6. Comparison of our limits with the latest constraints from Super-Kamiokande [2] and PICO [32, 33]. Depending on the annihilation channel, IceCube provides the strongest limits above WIMP masses of ∼100–200 GeV. Super-K is more sensitive at the lowest masses. If the annihilation spectrum is soft or heavily suppressed, the PICO experiment provides stronger limits than neutrino telescopes; other direct limits are weaker. Here we have assumed an annihilation cross-section of ⟨σv⟩0 = 3 × 10−26 cm3 s−1 for deriving IceCube limits; Super-K limits assume complete equilibrium between capture and annihilation in the Sun. 101 102 103 104 Dark matter mass mχ (GeV) 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 10−33 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 Super-K b¯b Super-K W +W − Super-K τ +τ − PICO-2L (2015) IceCube b¯b IceCube W +W − IceCube τ +τ − PICO-60 (2015) IceCube Collaboration 2016 JCAP04(2016)022 10 10 Dark matter mass mχ (GeV) Figure 6. Comparison of our limits with the latest constraints from Super-Kamiokande [2] and PICO [32, 33]. Depending on the annihilation channel, IceCube provides the strongest limits above WIMP masses of ∼100–200 GeV. Super-K is more sensitive at the lowest masses. If the annihilation spectrum is soft or heavily suppressed, the PICO experiment provides stronger limits than neutrino telescopes; other direct limits are weaker. Here we have assumed an annihilation cross-section of ⟨σv⟩0 = 3 × 10−26 cm3 s−1 for deriving IceCube limits; Super-K limits assume complete equilibrium between capture and annihilation in the Sun. pends on the annihilation channel). Super-Kamiokande [2] is the most sensitive experiment at all lower masses. Limits from direct detection [32, 33] are weaker, except in the case of DM with soft or suppressed annihilation spectra, in which case the PICO experiment [32, 33] is the most constraining. Indirect DM searches by Antares [35] and Baksan [36] have set less stringent limits on the spin-dependent DM-proton scattering and are consequently not included in ﬁgure 6. 4 Improved limits on WIMP dark matter Figure 7 shows new limits for all major two-body annihilation ﬁnal states. Annihilation to either electroweak gauge boson ﬁnal state is more or less equivalent, as W and Z have around the same mass and couplings to the rest of the SM, and consequently yield very similar neutrino spectra. We don’t show hZ, but we have checked that it indeed lies mid-way between hh and ZZ, as expected. As expected, most channels are indeed bracketed by the canonical ‘hard’ and ‘soft’ channels. The exceptions to this are gluon ﬁnal states, where spectra are especially soft and – 17 – 101 102 103 104 Dark matter mass mχ (GeV) 10−42 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 10−33 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 IC79 gg b¯b hh t¯t W +W − ZZ τ +τ − ν¯ν Figure 7. Limits on the spin-dependent WIMP-proton cross-section from IC79, for a range of diﬀerent annihilation ﬁnal states. The canonical hard (W +W −and τ +τ −) and soft (b¯b) channels bracket the possible limits for diﬀerent models reasonably well. More extreme channels (hardest: ν¯ν, softest: gg) less often found in SUSY can lead to even stronger or weaker constraints. For the ν¯ν channel we have assumed equal branching fractions for all three neutrino ﬂavours. The ability to easily and quickly compute full limits for any combination of ﬁnal states is a particular feature of the method and tools we present in this paper. As a convenience, dataﬁles for all curves in this ﬁgure are available precomputed in the nulike download.5 101 102 103 104 Dark matter mass mχ (GeV) 10−42 10−41 10−40 10−39 10−38 10−37 10−36 10−35 10−34 10−33 Dark matter-proton cross-section σSD,p (cm2) IceCube Collaboration 2016 IC79 gg b¯b hh t¯t W +W − ZZ τ +τ − ν¯ν JCAP04(2016)022 10 10 Dark matter mass mχ (GeV) Figure 7. Limits on the spin-dependent WIMP-proton cross-section from IC79, for a range of diﬀerent annihilation ﬁnal states. The canonical hard (W +W −and τ +τ −) and soft (b¯b) channels bracket the possible limits for diﬀerent models reasonably well. More extreme channels (hardest: ν¯ν, softest: gg) less often found in SUSY can lead to even stronger or weaker constraints. For the ν¯ν channel we have assumed equal branching fractions for all three neutrino ﬂavours. 4 Improved limits on WIMP dark matter The ability to easily and quickly compute full limits for any combination of ﬁnal states is a particular feature of the method and tools we present in this paper. As a convenience, dataﬁles for all curves in this ﬁgure are available precomputed in the nulike download.5 limits particularly weak, and neutrino ﬁnal states, which give very strong limits because they are monochromatic at the source. In the neutrino case, the monochromatic source spectrum means that of all ﬁnal states, annihilation to neutrinos tracks the actual neutrino eﬀective area most closely, with the only deviation from a monochromatic spectrum at the detector coming from reprocessing in the Sun following prompt production at the DM mass. This is also why the neutrino-channel limits at masses above one TeV become weaker than those from the τ +τ −channel: as a channel with an extremely hard annihilation spectrum, most of the neutrinos produced are close to the DM mass, and are therefore absorbed in the Sun. This is a general feature of all channels above one TeV: soft and hard channels begin to swap character in terms of the limits, as softer channels actually produce more neutrinos able to make it out of the Sun and to the detector. This eﬀect can also be seen in the gluon channel limits, which become stronger as the mass increases past ∼7 TeV, as enough of the limits particularly weak, and neutrino ﬁnal states, which give very strong limits because they are monochromatic at the source. In the neutrino case, the monochromatic source spectrum means that of all ﬁnal states, annihilation to neutrinos tracks the actual neutrino eﬀective area most closely, with the only deviation from a monochromatic spectrum at the detector coming from reprocessing in the Sun following prompt production at the DM mass. This is also why the neutrino-channel limits at masses above one TeV become weaker than those from the τ +τ −channel: as a channel with an extremely hard annihilation spectrum, most of the neutrinos produced are close to the DM mass, and are therefore absorbed in the Sun. This is a general feature of all channels above one TeV: soft and hard channels begin to swap character in terms of the limits, as softer channels actually produce more neutrinos able to make it out of the Sun and to the detector. 5 Implications for MSSM benchmarks In this section we use the new IceCube 79-string likelihood to test a number of models of weak-scale supersymmetry, employing the same test statistic as in section 4. Here we focus on the MSSM-25, a 25-parameter, weak-scale parameterisation of the minimal supersymmetric standard model (MSSM; see ref. [39] for details). This contains the MSSM-19, otherwise known to as the ‘phenomenological’ (p)MSSM, as a subspace. JCAP04(2016)022 Figure 8 shows some MSSM-25 benchmark models from the study of ref. [39], selected by requiring models with large spin-dependent scattering cross-sections. To give a broader indication of the possibilities in the MSSM, ﬁgure 8 also shows all models from the MSSM- 19 benchmarking exercise of the Snowmass 2013 review [9], except for the Bino-stop co- annihilation benchmark, which is very similar to the Bino-squark benchmark in this plane.6 Except for the models that we show with faded symbols (which we return to later) these models are all consistent with constraints from the LHC, ﬂavour physics and the relic density of dark matter, as well as direct and indirect searches for dark matter. The Snowmass 2013 benchmarks include a ‘spoke’ of models extending along a single direction in parameter space from one speciﬁc benchmark, shown as a vertical line in ﬁgure 8. We also show shaded bands between the strongest (τ +τ −) and weakest (b¯b) limits for channels typically seen in the MSSM. This gives some idea of where essentially all MSSM models are excluded regardless of annihilation channel (above the b¯b limit), and where only some models are excluded (between b¯b and τ +τ −), depending on their speciﬁc annihilation branching fractions to diﬀerent ﬁnal states. We have colour-coded the individual models in ﬁgure 8 by the extent to which they are excluded by the new IceCube limits, taking into account both spin-dependent and spin- independent scattering. We have also labelled diﬀerent benchmark groups according to the means by which the neutralino achieves the appropriate relic density in the early Universe. Many neutralino models are excluded for the ﬁrst time by the new limits we present here (bright red crosses). Other models exhibit a tension with data at the 68–90% conﬁdence level (orange crosses). These are ‘well-tempered’ neutralino models, which exhibit a roughly even mixture of gaugino and Higgsino weak eigenstates, boosting their spin-dependent scat- tering cross-section without contributing too strongly to the spin-independent one. 6We have reduced the Bino mass parameter M1 in this benchmark from 868 GeV to 800 GeV, in order to make the neutralino the lightest SUSY particle when carrying out the calculations with DarkSUSY. This reduces the nuclear scattering cross-sections compared to ref. [9], but the model is unconstrained by IceCube either way. 4 Improved limits on WIMP dark matter This eﬀect can also be seen in the gluon channel limits, which become stronger as the mass increases past ∼7 TeV, as enough of the – 18 – resulting very low-energy neutrinos are pulled into the observable energy window from below to counteract the slight increase in the number of neutrinos above one TeV that never make it out of the Sun. 5 Implications for MSSM benchmarks Other benchmarks (green plus symbols), where the relic density is achieved by squark or chargino co-annihilation with the neutralino, resonant annihilation via the CP-odd Higgs, or by virtue of the large annihilation cross-section exhibited by pure Higgsinos, remain unconstrained by spin-dependent searches of any kind. We also show a number of well-tempered neutralino benchmarks with faded symbols in ﬁgure 8, indicating that although they were consistent with all earlier data, they have since been excluded by LUX [38]. One of these examples (the well-tempered neutralino MSSM-19 benchmark from ref. [9]) was already strongly excluded by the original LUX spin- independent limits. The others satisfy the spin-independent limit, but are excluded by the – 19 – 101 102 103 104 Dark matter mass mχ (GeV) 10−45 10−44 10−43 10−42 10−41 10−40 10−39 10−38 10−37 10−36 10−35 Eﬀective χ0 1-proton cross-section σ′ SD,p (cm2) IceCube Collaboration 2016 Well-tempered neutralinos χ± coannihilation A funnel Pure ˜h ˜B −˜q coann. Area where essentially all MSSM models are excluded by IC79 MSSM excluded by IC79 depending on channel MSSM models not excluded by IC79 τ +τ − b¯b MSSM-25 benchmarks excluded at >90% CL tension (68% – 90% CL) allowed Figure 8. Implications of the new IC79 analysis for benchmark models in the MSSM-25. Models shown with solid red crosses are excluded for the ﬁrst time by IC79. Faded red symbols are excluded by both IC79 and recent LUX spin-dependent bounds [37, 38]. These all correspond to so-called ‘well-tempered’ neutralinos, which exhibit a mixed gaugino-Higgsino character. Solid orange crosses indicate models in tension with IC79 data at more than 1σ (but excluded at less than 90% CL). Green plus symbols indicate models not constrained by IC79, labelled according to the dominant characteristic determining their relic density. The vertical green line corresponds to a benchmark ‘spoke’ of models [9], where the correct relic density is obtained by bino-squark co-annihilation. Benchmarks are from the MSSM-25 and MSSM-19 (‘pMSSM’; a subset of the MSSM-25) scans of refs. [9, 39], and correspond to models allowed by LHC, relic density and other direct and indirect constraints. Benchmark scattering cross-sections are rescaled for the neutralino relic density, and the shaded regions are indicative only; these assume pure spin-dependent scattering and annihilation to the canonical ‘hard’ and ‘soft’ channels often seen in the MSSM (even though harder and softer spectra are also possible within the MSSM). 7We note that shortly before this paper was accepted, LUX submitted their own oﬃcial spin-dependent analysis [40], which improves on the LUXCalc limits. 5 Implications for MSSM benchmarks 101 102 103 104 Dark matter mass mχ (GeV) 10−45 10−44 10−43 10−42 10−41 10−40 10−39 10−38 10−37 10−36 10−35 Eﬀective χ0 1-proton cross-section σ′ SD,p (cm2) IceCube Collaboration 2016 Well-tempered neutralinos χ± coannihilation A funnel Pure ˜h ˜B −˜q coann. Area where essentially all MSSM models are excluded by IC79 MSSM excluded by IC79 depending on channel MSSM models not excluded by IC79 τ +τ − b¯b MSSM-25 benchmarks excluded at >90% CL tension (68% – 90% CL) allowed JCAP04(2016)022 Figure 8. Implications of the new IC79 analysis for benchmark models in the MSSM-25. Models shown with solid red crosses are excluded for the ﬁrst time by IC79. Faded red symbols are excluded by both IC79 and recent LUX spin-dependent bounds [37, 38]. These all correspond to so-called ‘well-tempered’ neutralinos, which exhibit a mixed gaugino-Higgsino character. Solid orange crosses indicate models in tension with IC79 data at more than 1σ (but excluded at less than 90% CL). Green plus symbols indicate models not constrained by IC79, labelled according to the dominant characteristic determining their relic density. The vertical green line corresponds to a benchmark ‘spoke’ of models [9], where the correct relic density is obtained by bino-squark co-annihilation. Benchmarks are from the MSSM-25 and MSSM-19 (‘pMSSM’; a subset of the MSSM-25) scans of refs. [9, 39], and correspond to models allowed by LHC, relic density and other direct and indirect constraints. Benchmark scattering cross-sections are rescaled for the neutralino relic density, and the shaded regions are indicative only; these assume pure spin-dependent scattering and annihilation to the canonical ‘hard’ and ‘soft’ channels often seen in the MSSM (even though harder and softer spectra are also possible within the MSSM). Figure 8. Implications of the new IC79 analysis for benchmark models in the MSSM-25. Models shown with solid red crosses are excluded for the ﬁrst time by IC79. Faded red symbols are excluded by both IC79 and recent LUX spin-dependent bounds [37, 38]. These all correspond to so-called ‘well-tempered’ neutralinos, which exhibit a mixed gaugino-Higgsino character. Solid orange crosses indicate models in tension with IC79 data at more than 1σ (but excluded at less than 90% CL). Green plus symbols indicate models not constrained by IC79, labelled according to the dominant characteristic determining their relic density. The vertical green line corresponds to a benchmark ‘spoke’ of models [9], where the correct relic density is obtained by bino-squark co-annihilation. 5 Implications for MSSM benchmarks Properties of the benchmark models shown in ﬁgure 8. mχ ⟨σv⟩ Ωh2 σSIp σSDp σ ′ SIp σ ′ SDp C A Dominant annihilation ﬁnal states Excl. 5 Implications for MSSM benchmarks (GeV) (cm−3 s−1) (cm2) (cm2) (cm2) (cm2) (s−1) (s−1) (contributions <1% not shown) (%CL) 67.8 4.2 ×10−28 0.123 4.1 ×10−47 1.6 ×10−39 4.1 ×10−47 1.6 ×10−39 7.9 ×1023 4.0 ×1023 τ +τ −(59%), b¯b (16%), c¯c (14%), gg (10%), Zγ (1%) 100.0 75.3 6.4 ×10−28 0.109 5.7 ×10−47 1.0 ×10−39 5.1 ×10−47 9.3 ×10−40 3.9 ×1023 2.0 ×1023 τ +τ −(71%), b¯b (14%), c¯c (6%), gg (5%), Zγ (3%), γγ (1%) 100.0 95.6 1.6 ×10−26 0.103 4.7 ×10−48 7.4 ×10−40 4.0 ×10−48 6.3 ×10−40 1.7 ×1023 8.4 ×1022 W +W −(85%), τ +τ −(12%), ZZ (3%) 99.4 102.9 1.9 ×10−26 0.115 5.8 ×10−46 2.0 ×10−40 5.5 ×10−46 1.9 ×10−40 4.5 ×1022 2.3 ×1022 τ +τ −(93%), W +W −(5%), ZZ (2%) 92.1 116.6 2.0 ×10−26 0.101 6.7 ×10−46 4.0 ×10−40 5.6 ×10−46 3.3 ×10−40 6.2 ×1022 3.1 ×1022 W +W −(53%), ZZ (30%), b¯b (14%), Zh (2%) 76.6 116.6 1.7 ×10−26 0.114 2.4 ×10−46 4.0 ×10−40 2.2 ×10−46 3.8 ×10−40 7.1 ×1022 3.5 ×1022 W +W −(62%), ZZ (35%), Zh (2%), b¯b (1%) 94.0 147.5 1.8 ×10−26 0.123 4.9 ×10−48 2.7 ×10−40 4.9 ×10−48 2.7 ×10−40 3.2 ×1022 1.6 ×1022 W +W −(52%), ZZ (36%), Zh (7%), b¯b (4%), τ +τ −(1%) 99.5 149.0 2.6 ×10−26 0.086 2.1 ×10−44 2.8 ×10−40 1.5 ×10−44 2.0 ×10−40 2.4 ×1022 1.2 ×1022 W +W −(53%), ZZ (37%), Zh (6%), b¯b (2%), τ +τ −(1%) 98.1 168.1 1.7 ×10−26 0.121 1.3 ×10−45 1.9 ×10−40 1.3 ×10−45 1.9 ×10−40 1.8 ×1022 9.0 ×1021 W +W −(51%), ZZ (39%), Zh (5%), b¯b (3%), τ +τ −(2%) 99.7 175.9 1.7 ×10−26 0.121 7.8 ×10−48 1.5 ×10−40 7.8 ×10−48 1.5 ×10−40 1.2 ×1022 6.2 ×1021 t¯t (52%), W +W −(25%), ZZ (19%), Zh (3%), gg (1%) 79.1 275.4 5.7 ×10−27 0.116 3.5 ×10−45 7.3 ×10−41 3.4 ×10−45 7.0 ×10−41 2.6 ×1021 1.3 ×1021 W +W −(46%), ZZ (37%), b¯b (12%), τ +τ −(3%), Zh (2%), 95.6 gg (1%) 799.4 2.9 ×10−29 0.046 8.0 ×10−47 1.4 ×10−43 3.0 ×10−47 5.2 ×10−44 3.9 ×1017 1.9 ×1014 gg (64%), τ +τ −(18%), b¯b (16%), γγ (2%) 0.0 971.6 1.4 ×10−26 0.100 5.2 ×10−46 3.4 ×10−43 4.4 ×10−46 2.9 ×10−43 2.5 ×1018 1.1 ×1018 W +W −(44%), ZZ (35%), W ±H∓(16%), A0h (3%), Zγ (1%) 5.2 1019.1 4.3 ×10−28 0.064 1.8 ×10−47 1.7 ×10−44 1.8 ×10−47 1.7 ×10−44 1.1 ×1017 3.0 ×1014 b¯b (69%), t¯t (17%), τ +τ −(13%), Zh (1%) 0.0 1031.3 1.0 ×10−26 0.106 9.7 ×10−47 7.9 ×10−44 8.6 ×10−47 7.0 ×10−44 4.7 ×1017 1.0 ×1017 W +W −(51%), ZZ (42%), W ±H∓(6%), A0h (1%), Zγ (1%) 0.0 1078.2 1.0 ×10−26 0.102 3.4 ×10−45 1.4 ×10−42 2.9 ×10−45 1.2 ×10−42 1.2 ×1019 6.0 ×1018 W +W −(51%), ZZ (37%), Zh (6%), t¯t (4%), Zγ (1%) 13.5 1157.6 1.2 ×10−26 0.109 1.2 ×10−45 2.5 ×10−43 1.1 ×10−45 2.2 ×10−43 3.4 ×1018 1.6 ×1018 W +W −(35%), ZZ (29%), W ±H∓(20%), A0h (5%), Zh (4%), 6.5 ZH (3%), A0H (3%), t¯t (1%) 1213.7 1.5 ×10−26 0.103 8.7 ×10−45 2.2 ×10−42 7.5 ×10−45 1.8 ×10−42 2.2 ×1019 1.1 ×1019 W +W −(30%), W ±H∓(27%), ZZ (20%), A0h (7%), t¯t (5%), 18.0 Zh (4%), A0H (3%), ZH (3%), b¯b (1%) Table 1. 5 Implications for MSSM benchmarks Benchmarks are from the MSSM-25 and MSSM-19 (‘pMSSM’; a subset of the MSSM-25) scans of refs. [9, 39], and correspond to models allowed by LHC, relic density and other direct and indirect constraints. Benchmark scattering cross-sections are rescaled for the neutralino relic density, and the shaded regions are indicative only; these assume pure spin-dependent scattering and annihilation to the canonical ‘hard’ and ‘soft’ channels often seen in the MSSM (even though harder and softer spectra are also possible within the MSSM). recent LUXCalc [37] application of the LUX data to spin-dependent neutron scattering.7 All of these models are strongly excluded by IceCube. recent LUXCalc [37] application of the LUX data to spin-dependent neutron scattering.7 All of these models are strongly excluded by IceCube. In table 1, we give further details of all the benchmark models shown in ﬁgure 8. These include cross-sections for annihilation and nuclear scattering (⟨σv⟩, σSD, σSI), relic densities – 20 – mχ ⟨σv⟩ Ωh2 σSIp σSDp σ ′ SIp σ ′ SDp C A Dominant annihilation ﬁnal states Excl. 5 Implications for MSSM benchmarks (GeV) (cm−3 s−1) (cm2) (cm2) (cm2) (cm2) (s−1) (s−1) (contributions <1% not shown) (%CL) 67.8 4.2 ×10−28 0.123 4.1 ×10−47 1.6 ×10−39 4.1 ×10−47 1.6 ×10−39 7.9 ×1023 4.0 ×1023 τ +τ −(59%), b¯b (16%), c¯c (14%), gg (10%), Zγ (1%) 100.0 75.3 6.4 ×10−28 0.109 5.7 ×10−47 1.0 ×10−39 5.1 ×10−47 9.3 ×10−40 3.9 ×1023 2.0 ×1023 τ +τ −(71%), b¯b (14%), c¯c (6%), gg (5%), Zγ (3%), γγ (1%) 100.0 95.6 1.6 ×10−26 0.103 4.7 ×10−48 7.4 ×10−40 4.0 ×10−48 6.3 ×10−40 1.7 ×1023 8.4 ×1022 W +W −(85%), τ +τ −(12%), ZZ (3%) 99.4 102.9 1.9 ×10−26 0.115 5.8 ×10−46 2.0 ×10−40 5.5 ×10−46 1.9 ×10−40 4.5 ×1022 2.3 ×1022 τ +τ −(93%), W +W −(5%), ZZ (2%) 92.1 116.6 2.0 ×10−26 0.101 6.7 ×10−46 4.0 ×10−40 5.6 ×10−46 3.3 ×10−40 6.2 ×1022 3.1 ×1022 W +W −(53%), ZZ (30%), b¯b (14%), Zh (2%) 76.6 116.6 1.7 ×10−26 0.114 2.4 ×10−46 4.0 ×10−40 2.2 ×10−46 3.8 ×10−40 7.1 ×1022 3.5 ×1022 W +W −(62%), ZZ (35%), Zh (2%), b¯b (1%) 94.0 147.5 1.8 ×10−26 0.123 4.9 ×10−48 2.7 ×10−40 4.9 ×10−48 2.7 ×10−40 3.2 ×1022 1.6 ×1022 W +W −(52%), ZZ (36%), Zh (7%), b¯b (4%), τ +τ −(1%) 99.5 149.0 2.6 ×10−26 0.086 2.1 ×10−44 2.8 ×10−40 1.5 ×10−44 2.0 ×10−40 2.4 ×1022 1.2 ×1022 W +W −(53%), ZZ (37%), Zh (6%), b¯b (2%), τ +τ −(1%) 98.1 168.1 1.7 ×10−26 0.121 1.3 ×10−45 1.9 ×10−40 1.3 ×10−45 1.9 ×10−40 1.8 ×1022 9.0 ×1021 W +W −(51%), ZZ (39%), Zh (5%), b¯b (3%), τ +τ −(2%) 99.7 175.9 1.7 ×10−26 0.121 7.8 ×10−48 1.5 ×10−40 7.8 ×10−48 1.5 ×10−40 1.2 ×1022 6.2 ×1021 t¯t (52%), W +W −(25%), ZZ (19%), Zh (3%), gg (1%) 79.1 275.4 5.7 ×10−27 0.116 3.5 ×10−45 7.3 ×10−41 3.4 ×10−45 7.0 ×10−41 2.6 ×1021 1.3 ×1021 W +W −(46%), ZZ (37%), b¯b (12%), τ +τ −(3%), Zh (2%), 95.6 gg (1%) 799.4 2.9 ×10−29 0.046 8.0 ×10−47 1.4 ×10−43 3.0 ×10−47 5.2 ×10−44 3.9 ×1017 1.9 ×1014 gg (64%), τ +τ −(18%), b¯b (16%), γγ (2%) 0.0 971.6 1.4 ×10−26 0.100 5.2 ×10−46 3.4 ×10−43 4.4 ×10−46 2.9 ×10−43 2.5 ×1018 1.1 ×1018 W +W −(44%), ZZ (35%), W ±H∓(16%), A0h (3%), Zγ (1%) 5.2 1019.1 4.3 ×10−28 0.064 1.8 ×10−47 1.7 ×10−44 1.8 ×10−47 1.7 ×10−44 1.1 ×1017 3.0 ×1014 b¯b (69%), t¯t (17%), τ +τ −(13%), Zh (1%) 0.0 1031.3 1.0 ×10−26 0.106 9.7 ×10−47 7.9 ×10−44 8.6 ×10−47 7.0 ×10−44 4.7 ×1017 1.0 ×1017 W +W −(51%), ZZ (42%), W ±H∓(6%), A0h (1%), Zγ (1%) 0.0 1078.2 1.0 ×10−26 0.102 3.4 ×10−45 1.4 ×10−42 2.9 ×10−45 1.2 ×10−42 1.2 ×1019 6.0 ×1018 W +W −(51%), ZZ (37%), Zh (6%), t¯t (4%), Zγ (1%) 13.5 1157.6 1.2 ×10−26 0.109 1.2 ×10−45 2.5 ×10−43 1.1 ×10−45 2.2 ×10−43 3.4 ×1018 1.6 ×1018 W +W −(35%), ZZ (29%), W ±H∓(20%), A0h (5%), Zh (4%), 6.5 ZH (3%), A0H (3%), t¯t (1%) 1213.7 1.5 ×10−26 0.103 8.7 ×10−45 2.2 ×10−42 7.5 ×10−45 1.8 ×10−42 2.2 ×1019 1.1 ×1019 W +W −(30%), W ±H∓(27%), ZZ (20%), A0h (7%), t¯t (5%), 18.0 Zh (4%), A0H (3%), ZH (3%), b¯b (1%) Table 1. 6 Conclusions JCAP04(2016)022 We have presented a new analysis of data collected in the 79-string IceCube search for dark matter, taking into account energies of individual neutrino events. This resulted in stronger spin-dependent limits on WIMP dark matter, in particular for high WIMP masses, and allowed us to rule out a number of MSSM models for the ﬁrst time. In the process, we developed an updated fast likelihood pipeline for event-level neutrino telescope DM search data, allowing it to be quickly and accurately applied to constrain essentially any dark matter model. We have also provided a public code implementing the new likelihood (nulike), and made data from the 79-string IceCube DM search publicly available in a format compatible with its use. Full details of the SUSY benchmarks and generic WIMP results presented in this paper are available as example programs in the public distribution of nulike. 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Acknowledgments We acknowledge the support from the following agencies: U.S. National Science Foundation- Oﬃce of Polar Programs, U.S. National Science Foundation-Physics Division, University of Wisconsin Alumni Research Foundation, the Grid Laboratory Of Wisconsin (GLOW) grid infrastructure at the University of Wisconsin — Madison, the Open Science Grid (OSG) grid infrastructure; U.S. Department of Energy, and National Energy Research Scientiﬁc Comput- ing Center, the Louisiana Optical Network Initiative (LONI) grid computing resources; Nat- ural Sciences and Engineering Research Council of Canada, WestGrid and Compute/Calcul Canada; Swedish Research Council, Swedish Polar Research Secretariat, Swedish National Infrastructure for Computing (SNIC), and Knut and Alice Wallenberg Foundation, Swe- den; German Ministry for Education and Research (BMBF), Deutsche Forschungsgemein- schaft (DFG), Helmholtz Alliance for Astroparticle Physics (HAP), Research Department of Plasmas with Complex Interactions (Bochum), Germany; Fund for Scientiﬁc Research (FNRS-FWO), FWO Odysseus programme, Flanders Institute to encourage scientiﬁc and technological research in industry (IWT), Belgian Federal Science Policy Oﬃce (Belspo); University of Oxford, United Kingdom; Marsden Fund, New Zealand; Australian Research Council; Japan Society for Promotion of Science (JSPS); the Swiss National Science Founda- tion (SNSF), Switzerland; National Research Foundation of Korea (NRF); Danish National Research Foundation, Denmark (DNRF); Science and Technology Facilities Council, United Kingdom (STFC). 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https://openalex.org/W2045546296	https://europepmc.org/articles/pmc4324074?pdf=render	English	null	A method for a categorized and probabilistic analysis of the surface electromyogram in dynamic contractions	Frontiers in physiology	2,015	cc-by	7,439	Correspondence: Correspondence: Sylvie C. F. A. Von Werder, Department of Rehabilitation & Prevention Engineering, Institute of Applied Medical Engineering, Helmholtz Institute, RWTH Aachen University, Pauwelsstraße 20, 52074 Aachen, Germany e-mail: vonwerder@ hia.rwth-aachen.de Correspondence: Sylvie C. F. A. Von Werder, Department of Rehabilitation & Prevention Engineering, Institute of Applied Medical Engineering, Helmholtz Institute, RWTH Aachen University, Pauwelsstraße 20, 52074 Aachen, Germany e-mail: vonwerder@ hia.rwth-aachen.de INTRODUCTION they noted the shape and thickness of the investigated muscle and surrounding tissue, the electrode position and inter-electrode dis- tance, crosstalk of nearby muscles, as well as ﬁber membrane and motor unit properties, which can affect the recording and conse- quently the interpretation of the sEMG. Furthermore, individual differences due to the muscular condition or habitual motion pat- terns might affect the sEMG and contribute to the challenge of interpreting dynamic sEMG recordings. Muscles move us and many scientists move the research on the interpretation of muscular activation. Thus, muscular activation is investigated and described in a wealth of literature to increase the knowledge of physiological movement patterns or pathologi- cal movement disorders. But, the human motor system permits a wide variety of complex movements. To meet the challenge of controlling all possible movements, the human motor sys- tem exhibits redundant muscular activation strategies ranging from strategies on how to activate one muscle to strategies on activating a consortium of contributing muscles. The study of muscular activation with surface Electromyography (sEMG) pro- vides insights into the contribution of single muscles to the overall performed movement. Like the human motor system itself, the interpretation of muscular activation is complex. There are con- troversial results from examination of sEMG as well as reviews on the limitation of sEMG. The interpretation of sEMG record- ings requires caution (Rau et al., 2004) especially in dynamic tasks (Farina, 2006). The sEMG is dependent on several factors, which impact on the relation between the recorded sEMG and the underlying physiological process. Farina et al. divided the fac- tors that inﬂuence the sEMG in two groups, “non-physiological” and “physiological” factors (Farina et al., 2004). Amongst others, p g y g But, especially dynamic tasks are of high importance for con- siderations on pathological movement disorders. At this point, daily living activities which involve free movements of the limbs are restricted. On the one hand, analyzing free movements fur- ther challenges the interpretation of the dynamically recorded sEMG. ORIGINAL RESEARCH ARTICLE bli h d 11 F b 2015 published: 11 February 2015 doi: 10.3389/fphys.2015.00030 Sylvie C. F. A. Von Werder 1, Tim Kleiber 1,2 and Catherine Disselhorst-Klug 1 1 Department of Rehabilitation & Prevention Engineering, Institute of Applied Medical Engineering, RWTH Aachen University, Aachen, Germany 2 German Research School for Simulation Sciences, Joint Graduate School of RWTH Aachen University and Forschungszentrum Jülich, Jülich, Germany The human motor system permits a wide variety of complex movements. Thereby, the inter-individual variability as well as the biomechanical aspects of the performed movement itself contribute to the challenge of the interpretation of sEMG signals in dynamic contractions. A procedure for the systematic analysis of sEMG recordings during dynamic contraction was introduced, which includes categorization of the data in combination with the analysis of frequency distributions of the sEMG with a probabilistic approach. Using the example of elbow ﬂexion and extension the procedure was evaluated with 10 healthy subjects. The recorded sEMG signals of brachioradialis were categorized into a combination of constant and variable movement factors, which originate from the performed movement. Subsequently, for each combination of movement factors cumulative frequency distributions were computed for each subject separately. Finally, the probability of the difference of muscular activation in varying movement conditions was assessed. The probabilistic approach was compared to a deterministic analysis of the same data. Both approaches observed a signiﬁcant change of muscular activation of brachioradialis during concentric and eccentric contractions exclusively for ﬂexion and extension angles exceeding 30◦. However, with the probabilistic approach additional information on the likelihood that the tested effect occurs can be provided. Especially for movements under uncontrollable boundary conditions, this information to assess the conﬁdence of the detected results is of high relevance. Thus, the procedure provides new insights into the quantiﬁcation and interpretation of muscular activity. Keywords: sEMG, dynamic, probabilistic, categorization, contraction type, joint angle Edited by: Edited by: Taian Mello Martins Vieira, Universidade Federal do Rio de Janeiro, Brazil Reviewed by: Marcos Duarte, Federal University of ABC, Brazil Alessio Gallina, University of British Columbia, Canada Correspondence: Sylvie C. F. A. Von Werder, Department of Rehabilitation & Prevention Engineering, Institute of Applied Medical Engineering, Helmholtz Institute RWTH Aachen Taian Mello Martins Vieira, Universidade Federal do Rio de Janeiro, Brazil Reviewed by: Marcos Duarte, Federal University of ABC, Brazil Alessio Gallina, University of British Columbia, Canada Reviewed by: Marcos Duarte, Federal University of ABC, Brazil Alessio Gallina, University of British Columbia, Canada A method for a categorized and probabilistic analysis of the surface electromyogram in dynamic contractions Sylvie C. F. A. Von Werder 1, Tim Kleiber 1,2 and Catherine Disselhorst-Klug 1 Correspondence: Sylvie C. F. A. Von Werder, Department of Rehabilitation & Prevention Engineering, Institute of Applied Medical Engineering, Helmholtz Institute, RWTH Aachen University, Pauwelsstraße 20, 52074 Aachen, Germany e-mail: vonwerder@ hia.rwth-aachen.de CONCEPT OF SYSTEMATIC ANALYSIS Before analyzing the muscular activation in dynamic contraction possible inﬂuencing factors of the intended movement must be extracted. In the case of elbow ﬂexion and extension, several fac- tors which inﬂuence the muscular activation and origin from the type of the performed movement are known. These factors include the impact of the joint position, meaning the supinated, neutral or pronated forearm position, or the impact of concentric or eccentric contraction types. Furthermore, the external force as well as the velocity of the performed movement might impact on the muscular activation. Each movement factor must be assigned to remain either constant or variable during the intended move- ment of the study. If one factor remains constant during the experiment, the interpretation of the data is limited to this con- stant factor and consequently all constant movement factors must be indicated. Regarding the impact of the performed movement itself, sev- eral factors have been described which inﬂuence the muscular activation and originate from the way in which the performed movement was conducted. Considering the elbow ﬂexion and extension movement, the effect of muscular contraction type on the activation of contributing elbow muscles was evaluated. In that context, a decreased activation during eccentric contrac- tion depending on the joint angle and pre-activation mode was observed (Komi et al., 2000; Linnamo et al., 2006). Additionally, differences of angle dependent variations of the synergistic acti- vation of biceps brachii and brachioradialis during eccentric contractions were found, which were not observed during con- centric contractions (Nakazawa et al., 1993). Besides the per- formed movement itself, there is an inter-individual variability of sEMG proﬁles (Guidetti et al., 1996; Hug, 2011). As a result the data can be recorded in deﬁned constant or variable conditions of inﬂuencing factors, which originate from the performed movement. Subsequently, the raw data is processed and categorized into a combination of inﬂuencing movement fac- tors. For each categorized data a relative frequency distribution is computed to assign probabilities of the muscular activation in deﬁned dynamic movement conditions, which are further assessed in a probabilistic manner. Regarding the procedure of the performed analysis, the way of the interpretation of the processed data is crucial. Once the data is processed it can be assessed with a deterministic approach or with a probabilistic approach. PARTICIPANTS The experiments were conducted with 10 healthy subjects (age 23 ± 3 years; height 179 ± 9 cm; body mass 75 ± 8 kg). No subject had any known symptoms of neuromuscular disorders, history of orthopedic surgery or any affection of the upper extremities. Subjects avoided strenuous exercises in the day prior to the mea- surement. All subjects were right handed. All experiments were performed in accordance to the declaration of Helsinki and all subjects gave written informed consent prior to the study. CONCEPT OF SYSTEMATIC ANALYSIS For a deterministic approach the data is successively examined for each time point, to compute mean val- ues for the performed movement cycles as it was performed for the muscular activation patterns of the gait analysis (Perry, 1992). For a deterministic approach, the processed sEMG is utilized as input to compute for a ﬁx output, e.g., the mean ± standard deviation. In contrast, for a probabilistic approach, probabili- ties are assigned to the occurrence of speciﬁc events. Instead of comparing sEMG means ± standard deviation, a probabilistic approach assesses frequency distributions of the sEMG record- ings to compute probabilities that the analyzed sEMG recording takes a speciﬁc value. In that way the examined signal, with an uncertain nature of the sEMG recording itself is assessed with a fuzzy approach instead of crisp deterministic values. A proba- bilistic method was described to estimate the levels of muscular activation for the functional electrical stimulation (Anderson and Fuglevand, 2008) and the control of neuroprosthetics (Johnson and Fuglevand, 2009). Frontiers in Physiology \| Integrative Physiology Correspondence: If one factor experiment, the interpretation of stant factor and consequently all co be indicated. As a result the data can be re variable conditions of inﬂuencing he performed movement. Subsequ and categorized into a combinatio ors. For each categorized data a s computed to assign probabilit n deﬁned dynamic movement assessed in a probabilistic manner PARTICIPANTS Th i t d t d The procedure to systematically analyze sEMG recordings of dynamic contractions was introduced using the example of elbow ﬂexion and extension with respect to the muscular activation of brachioradialis. *Correspondence: On the other hand, possible sEMG ﬁndings under highly controlled movement conditions in a physiological reference can- not be easily transferred to the pathological situation of patients. Sometimes the patients are not able to move their limbs under the same highly controlled conditions, e.g., patients with spas- ticity after stroke. Thus, the balance between investigating freely performed movements on the one hand and the possibility to interpret the dynamic recorded sEMG on the other hand is intended. Considering the example of spasticity, the muscu- lar activation can be exclusively recorded in the range of the February 2015 \| Volume 6 \| Article 30 \| 1 www.frontiersin.org Probabilistic analysis of categorized sEMG Von Werder et al. patients’ movement capability, leading to uncontrollable bound- ary conditions of the investigated movement. Consequently, the performed movement is more complex just as the interpreta- tion of the sEMG. However, the interplay of the mechanisms, which contribute to the human motion, is to the same extent of physiological relevance as the controllable single physiological mechanisms themselves. Especially if pathologies are involved, the interplay of several factors is of high relevance. recorded data combined with a probabilistic approach. In that way the described procedure aims to account for the impact of the performed movement on the one hand and moreover aims to add an uncertain component to the interpretation of the recorded sEMG on the other hand to provide information on the con- ﬁdence of the obtained results. The probabilistic approach was compared to a deterministic analysis of the same data. MATERIALS AND METHODS If less restricted and uncontrollable movement conditions are inevitable, speciﬁc analyzing techniques are needed to assess the conﬁdence of the detected results. In this context is it possible to provide additional information on the likelihood of the occur- rence or absence of an investigated physiological or pathological effect? The procedure to systematically dynamic contractions was introdu ﬂexion and extension with respec brachioradialis. CONCEPT OF SYSTEMATIC ANALYSIS Before analyzing the muscular act possible inﬂuencing factors of the extracted. In the case of elbow ﬂex ors which inﬂuence the muscul he type of the performed movem nclude the impact of the joint po neutral or pronated forearm positi or eccentric contraction types. Fur well as the velocity of the perform he muscular activation. Each mov o remain either constant or varia ment of the study. CATEGORIZATION OF THE PROCESSED DATA A decision tree was used to distinguish the present combina- tion of variable movement factors for each sampling point of the recorded sEMG data. When the decision algorithm computes the combination of variable movement parameters, the amplitude of the enveloped and normalized sEMG value is stored with an assignment to this combination (Figure 1). Regarding the inﬂuencing movement factors, the joint posi- tion, the external force and the performed velocity were kept constant. The subjects were measured in standing position with anatomical hand posture, representing a neutral forearm posi- tion, with the shoulder in neutral rest position, meaning being neither ﬂexed nor abducted. The subjects had to ﬂex and extend their elbow with an external weight of 3.5 kg and a constant speed of 25◦/s. The elbow ﬂexion and extension angle as well as the contraction type were assigned as variable inﬂuencing movement factors. At this point, it must be marked that although the weight that the subject had to carry was constant at 3.5 kg, the exter- nal moment due to this weight changed with the ﬂexion and extension angles. Therefore, the angle impact is dependent on the amount of force produced to move the constant external weight. In the example of ﬂexion and extension of the elbow, combi- nations of the contraction type and ﬂexion and extension angle were assigned to the normalized sEMG value. First, the deci- sion algorithm derived the concentric or eccentric contraction type for the investigated sampling point. Thereby, the contrac- tion types were distinguished in two categories, concentric and eccentric contractions, which were derived from the slope of the ﬂexion and extension course. For the ﬂexing muscles, positive slopes representing ﬂexion of the joint were assigned to concen- tric contractions, whereas negative slopes representing extension of the joint were assigned to eccentric contractions. Subsequently, the decision algorithm assigned the ﬂexion and extension angle at this sampling point to an angle interval. Here, the decision tree merged the angles into 12 intervals with 10◦steps ranging from 0 to 120◦, where 0◦was full extension. In order to prevent fatigue, measuring intervals were limited to one repetition of ﬂexion and extension followed by 60 s of relaxation time. A minimum of 20 repetitions of full ﬂexion and extension movements of the elbow joint were performed to gather enough data for the subsequent probabilistic analysis (Anderson and Fuglevand, 2008; Johnson and Fuglevand, 2009). EXPERIMENTAL SETUP The experimental setup included the measurement of bipolar sEMG and elbow joint angles. sEMG-electrodes, with an inter- electrode distance of 2 cm (Hermens et al., 2000), were placed on the brachioradialis. For the placement of the electrodes the lower arm was moved to neutral forearm position. The subject was asked to perform an elbow ﬂexion against resistance at the wrist. The electrodes were placed at the level of one third from the fossa Taking the example of ﬂexion and extensions of the elbow, this report describes a procedure to analyze sEMG recordings of dynamic contractions with a systematic categorization of the February 2015 \| Volume 6 \| Article 30 \| 2 Frontiers in Physiology \| Integrative Physiology Probabilistic analysis of categorized sEMG Von Werder et al. cubit along the palpable muscle belly. sEMG signals were sam- pled at a frequency of 2000 Hz. Kinematics of elbow ﬂexion and extension movement were recorded with a VICON MX (10 cam- eras) motion capture system with a sampling rate of 200 Hz and an electrogoniometer (Biometrics Ltd) with a sampling frequency of 2000 Hz. The marker setup included ﬁve joint markers and four triplets of segment markers. The joint markers were placed at the radial and ulnar styloid process of the wrist, the epicondyle lateral and medial at the elbow and the acromion. The segment markers were placed at the hand, forearm, upper arm and thorax. ﬁve MVC trials with constant torques the mean of the three MVC sEMG envelopes with the smallest standard deviation is chosen as reference. FREQUENCY DISTRIBUTION FOR PROBABILISTIC ANALYSIS The impact of the variable movement factors of ﬂexion and exten- sion angle and contraction type for the combination of constant FIGURE 1 \| A decision tree is used to categorize the processed sEMG data into a combination of constant and variable movement factors to ﬁnally analyze relative frequency distributions of the sEMG with respect to all possible movement factors. CATEGORIZATION OF THE PROCESSED DATA The ﬂexion and extension course recorded with the electrogoniometer was visualized to provide a feedback on the constant velocity perfor- mance of the subjects. For normalization a set of ﬁve maximum voluntary contractions (MVC) in 90◦of elbow ﬂexion and neu- tral forearm position were performed prior to the elbow ﬂexion and extension measurements. Between each MVC trial a break of a minimum of 180 s was made to avoid the possibility of fatigue. The categorized data allocates the muscular activation to each combination of variable movement factors and was further assessed with a probabilistic approach. www.frontiersin.org SIGNAL PROCESSING Signal processing of the kinematic data included computation of ﬂexion and extension as well as pronation and supination angles. The marker trajectories were ﬁltered with a spline ﬁlter of third order (Schmidt et al., 1999). With the help of a biomechanical model of the human arm the pronation and supination angles of the elbow were computed (Rau et al., 2000; Williams et al., 2006). All data sets with a change of pronation and supination angles exceeding ±5◦were excluded from further analysis to ensure that the subjects remained their constant joint position and avoided any pro- or supinating movement of the elbow. Likewise, any data sets with movement of the adjacent joints, namely the shoulder and wrist, exceeding ±10◦were excluded. The sEMG recordings were band pass-ﬁltered (10–500 Hz), rectiﬁed and smoothed (moving average ﬁlter, window length 100 ms) in order to compute the envelope of the sEMG signal. Normalization of the envelope was performed with respect to the MVC. For that purpose the mean value of the enveloped sEMG of the recorded three MVC trials was computed. Out of FIGURE 1 \| A decision tree is used to categorize the processed sEMG data into a combination of constant and variable movement factors to ﬁnally analyze relative frequency distributions of the sEMG with respect to all possible movement factors. February 2015 \| Volume 6 \| Article 30 \| 3 www.frontiersin.org Probabilistic analysis of categorized sEMG Von Werder et al. the impact of movement factors on the muscular activation. Thus, the categorized frequency distributions of the normalized sEMG envelope were used for further analysis. To demonstrate the procedure, the following hypothesis was exemplarily tested. movement factors was further assessed with the help of a proba- bilistic approach. All amplitudes of the enveloped and normalized sEMG in one combination of angle category and contraction type were evaluated to derive a relative frequency distribution for each subject separately. From that relative frequency distribution a cumulative frequency can be computed to derive the probabil- ity for each possible sEMG value to be within a deﬁned interval (Figure 2). The muscular activation is affected by a combination of con- traction type and ﬂexion and extension angle. For the test of the raised hypothesis, the frequency distribu- tions of normalized sEMG envelopes in concentric and eccentric contraction were assessed in the same four ﬂexion and extension angle intervals as performed in analyzing scenario C. SIGNAL PROCESSING For that pur- pose a probability for each angle interval was computed for each subject as described in the following (Figure 3). EVALUATION OF CATEGORIZED DATA ANALYSIS To demonstrate the relevance of assigning possible movement parameters to the sEMG signals, the recorded sEMG signals of brachioradialis of all trials and all subjects were analyzed in three different analyzing scenarios in a deterministic way. For that pur- pose, for the same data the normalized sEMG envelopes were categorized with three different decision trees to compute mean values ± standard deviations of all trials and all subjects. The results were further analyzed for statistical relevance. For each subject the distance d between the most probable normalized sEMG envelope of the frequency distributions dur- ing concentric and eccentric contractions were computed for each angle interval separately. In the middle between the most proba- ble normalized sEMG envelope of the frequency distributions of concentric and eccentric contractions d/2 an analyzing interval Ainterval was deﬁned. The ﬁrst analyzing scenario A included a decision tree with the contraction type as variable movement parameter only, with- out any respect to the different elbow ﬂexion and extension angles. Exclusively the contraction type was used to categorize the processed sEMG signal into two contraction types. Ainterval = d 2 ± 2.5% MVC The second analyzing scenario B included a decision tree with the variable movement parameter ﬂexion and extension angles without any respect to the contraction type. Exclusively the ﬂex- ion and extension angles were used to categorize the processed sEMG into 12 intervals ranging from 0 to 120◦. For the statistical analysis the angle intervals were merged into four intervals with four times 30◦steps. Subsequently, the probability of the normalized sEMG envelope to occur within the deﬁned analyzing interval was computed for both contraction types separately, as concentric Pcon (Ainterval) and eccentric Pecc (Ainterval) cumulative frequencies. The probability that the value of normalized sEMG enve- lope of concentric and eccentric contractions occurs in both frequency distributions at the deﬁned interval Pcon + ecc (Ainterval) was computed through the multiplication of both cumulative frequencies: The third analyzing scenario C included the described decision tree with the combination of two variable movement parameters namely the combination of 12 angle and two contraction type intervals. For statistical analysis angle intervals were merged into four intervals with four times 30◦steps. Pcon + ecc (Ainterval) = Pcon (Ainterval) · Pecc (Ainterval) STATISTICAL ANALYSIS All statistical analyses were performed with SPSS (SPSS Inc., Chicago, IL, USA). The level of statistical signiﬁcance for all analyses was set to α = 0.05. First, the effect of categorization of the data was tested for the described analyzing scenarios. For the ﬁrst analyzing sce- nario A the contraction type was found to take a signiﬁcant impact on the muscular activation of brachioradialis. A statisti- cally signiﬁcant difference between normalized sEMG envelope of concentric contractions compared to eccentric contractions were found [t(9) = 5.256, p = 0.001] with a higher normalized sEMG during concentric contractions (19.14 ± 7.30% MVC) compared to eccentric contractions (13.76 ± 5.62% MVC). The difference of the concentric and eccentric normalized sEMG envelopes of the categorized data was normally distributed, as assessed by Shapiro-Wilk’s test (p = 0.703). For the categorized data of scenario A a paired-samples t-test was used to determine whether there was a statistically signiﬁcant difference between the categorized normalized sEMG envelopes (dependent variable) during concentric contractions compared to eccentric contractions. Test for normality was performed with Shapiro-Wilk’s test. For scenario B an One-Way repeated measures ANOVA was conducted to determine whether there were statistically signiﬁ- cant differences of the normalized sEMG envelope (dependent variable) over the course of four different angle conditions (within subject factor). A Post-hoc analysis with a Bonferroni adjustment was used to compare the change of the normalized sEMG envelope with increasing angles. Normal distribution and sphericity of the categorized data was assessed by Shapiro-Wilk’s test and Mauchly’s test of sphericity, respectively. For the second analyzing scenario B the ﬂexion and extension angle showed a signiﬁcant impact on the normalized sEMG enve- lope. The categorized normalized sEMG envelopes of scenario B were normally distributed at each angle condition, as assessed by Shapiro-Wilk’s test (p > 0.05). Mauchly’s test of sphericity indi- cated that the assumption of sphericity in test scenario B had been violated, χ2 = 20.489, p = 0.001. A Greenhouse-Geisser correc- tion (ε = 0.401) (Greenhouse and Geisser, 1959) was applied to correct the One-Way repeated measures ANOVA. The differ- ent angle conditions elicited statistically signiﬁcant changes of the mean normalized sEMG envelope [F(1.204, 9.633) = 39.793, For scenario C a Two-Way repeated measures ANOVA was run to determine the effect of four different angle conditions (within subject factor 1) in two different contraction types (within subject factor 2) on the normalized sEMG envelope (dependent variable). PROBABILISTIC ANALYSIS OF THE CATEGORIZED DATA The more the frequency distributions of concentric and eccentric contraction apart from each other, the smaller the probability for an overlap of both distributions at the deﬁned interval gets. The more the frequency distributions of concentric and eccentric contraction apart from each other, the smaller the probability for an overlap of both distributions at the deﬁned interval gets. The assignment of probabilities for each possible categorized sEMG value to be within a deﬁned interval enables the test of FIGURE 2 \| For each sampling point the value of the sEMG envelope is assigned to a combination of variable movement parameters. A relative frequency distribution of the corresponding sEMG is computed for each category of contraction type and ﬂexion and extension angle. Additionally, the probability of sEMG envelope to be within a deﬁned interval can be computed. category of contraction type and ﬂexion and extension angle. Additionally, the probability of sEMG envelope to be within a deﬁned interval can be computed. FIGURE 2 \| For each sampling point the value of the sEMG envelope is assigned to a combination of variable movement parameters. A relative frequency distribution of the corresponding sEMG is computed for each category of contraction type and ﬂexion and extension angle. Additionally, the probability of sEMG envelope to be within a deﬁned interval can be computed. February 2015 \| Volume 6 \| Article 30 \| 4 Frontiers in Physiology \| Integrative Physiology Frontiers in Physiology \| Integrative Physiology Probabilistic analysis of categorized sEMG Von Werder et al. FIGURE 3 \| Two examples for the procedure on the evaluation of two frequency distributions. The probability of overlap of concentric and eccentric frequency distributions of the same angle interval can be computed through the multiplication of the relative frequencies of each distribution to contain sEMG values in a deﬁned analyzing interval. This interval is located at d/2 in the middle between the most probable normalized sEMG envelopes of each distribution. If two distributions distinguish from each other, the probability of overlap would be zero. FIGURE 3 \| Two examples for the procedure on the evaluation of two frequency distributions. The probability of overlap of concentric and eccentric frequency distributions of the same angle interval can be computed through the multiplication of the relative frequencies of each distribution to contain sEMG values in a deﬁned analyzing interval. RESULTS The described method for the analysis of dynamic sEMG signals assigns the muscular activation to movement parameters origi- nating from the way of the performed movement. Thereby, the results are limited to the constant movement parameters, the constant velocity of 25◦/s and the constant external weight of 3.5 kg. Data are mean ± standard deviation, unless otherwise stated. www.frontiersin.org PROBABILISTIC ANALYSIS OF THE CATEGORIZED DATA This interval is located at d/2 in the middle between the most probable normalized sEMG envelopes of each distribution. If two distributions distinguish from each other, the probability of overlap would be zero. data was assessed by Shapiro-Wilk’s test and Mauchly’s test of sphericity, respectively. To compare the probabilistic approach to a deterministic anal- ysis the probabilities for the overlap of concentric and eccentric contractions in all four angle intervals of each subject were com- puted. Subsequently, the probabilities of each subject in one angle interval were averaged and ﬁnally compared to the deterministic outcome of the same categorized data of test scenario C. STATISTICAL ANALYSIS Subsequently, simple main effects on the contraction type were run to assess the difference of normalized sEMG in concentric and eccentric contractions under all angle conditions separately. Again, normal distribution and sphericity of the categorized February 2015 \| Volume 6 \| Article 30 \| 5 Probabilistic analysis of categorized sEMG Von Werder et al. p < 0.0005] with the normalized sEMG envelope increasing from 4.67 ± 2.28% MVC for ﬂexion angles of 0–30◦to 23.90 ± 10.55% MVC for ﬂexion angles of 90–120◦. Post-hoc analysis with a Bonferroni adjustment revealed that there was a statistically signiﬁcantly increase from 0–30◦to 30–60◦(p < 0.0005), from 30–60◦to 60–90◦(p = 0.005), but not for 60–90◦to 90–120◦ (p = 0.485). p < 0.0005] with the normalized sEMG envelope increasing from 4.67 ± 2.28% MVC for ﬂexion angles of 0–30◦to 23.90 ± 10.55% MVC for ﬂexion angles of 90–120◦. Post-hoc analysis with a Bonferroni adjustment revealed that there was a statistically signiﬁcantly increase from 0–30◦to 30–60◦(p < 0.0005), from 30–60◦to 60–90◦(p = 0.005), but not for 60–90◦to 90–120◦ (p = 0.485). The probabilistic analysis of scenario C supported that the combination of both movement parameters impacts on the mus- cular activation. Exemplarily Figure 4 illustrates the frequency distribution of the categorized sEMG of brachioradialis of one subject. The combination of contraction type and ﬂexion and extension angle can be assigned to a change of the processed sEMG signal. For small ﬂexion and extension angle no dif- ference of the most probable normalized sEMG envelope for eccentric and concentric contractions was found. With increasing angles the most probable normalized sEMG envelope was found to be higher in concentric contraction compared to eccentric contractions. Analysis of the studentized residuals of the categorized nor- malized sEMG envelopes of scenario C showed that there was normal distribution, as assessed by the Shapiro-Wilk test (p > 0.05). Additionally, there was sphericity for the interaction term, as assessed by Mauchly’s test of sphericity (p > 0.05). A sta- tistically signiﬁcant interaction between contraction type and angle condition was found [F(3, 27) = 24.732, p < 0.0005]. STATISTICAL ANALYSIS It is reasonable to assign possible ﬁnd- ings to a combination of variable and constant movement factors. But it is essential to highlight the constant movement factors, since the possible ﬁndings are limited to them. The deterministic as well as the probabilistic approach observed a signiﬁcant change of muscular activation of brachio- radialis during concentric and eccentric contractions exclusively for ﬂexion and extension angles exceeding 30◦(Table 1). muscular activation of brac ccentric contractions exclusi exceeding 30◦(Table 1). ic contractions contains mult ysis of the data and conseque etations. Sometimes the ana ynamic and complex movem ular if pathological movem ss restricted and uncontroll able and lead to a more com r the systematic analysis of sE raction includes a categoriza nalysis of frequency distribut nistic or probabilistic analysi tep must be assigned to a syst hrough the categorization of ree, the possible ﬁndings on m igned to a combination of mo lyzing scenarios revealed tha nsion of the elbow, the musc fected by the variable movem xion and extension angle. F n conclude that concentric c ized sEMG envelope amplit itionally, from the analyzing e amplitude of normalized sE n and extension angle. Howe that not the assignment of t the combination of both mo never changes in the musc p g For the comparison of the deterministic approach (Figure 5) to the probabilistic approach (Figure 6), summarized in Table 1, it can be concluded that both approaches reveal the same effect in that the concentric and eccentric activation of brachioradialis dif- fer from each other for angles exceeding 30◦. However, whereas the deterministic analysis computes for a statistical signiﬁcant effect, the probabilistic approach provides additional information on the probability that the same effect does or does not occur. The result from the repeated two way measure indicate a sta- tistical difference between concentric and eccentric contraction for angles exceeding 30◦, (p = 0.002, p = 0.001 and p < 0.0005 for angles between 30–60◦, 60–90◦, and 90–120◦, respectively). In contrast, the probabilistic parameter for the overlap of both frequency distributions provides additional information on the likelihood that this difference is not present. The decreased prob- abilities for angles exceeding 30◦represent that the frequency distributions of concentric and eccentric contractions are more likely to differ from each other. STATISTICAL ANALYSIS The simple main effects on the contraction type revealed that the normalized sEMG envelope was statistically signiﬁcantly differ- ent for the concentric compared to the eccentric contractions at the angle condition of angles between 30 and 60◦[F(1, 9) = 21.743, p = 0.002], 60–90◦[F(1, 9) = 27.803, p = 0.001], and 90–120◦[F(1, 9) = 36.070, p < 0.0005]. However, the normal- ized sEMG envelope was not statistically signiﬁcantly different for the concentric compared to the eccentric contractions at the angle condition of angles between 0 and 30◦[F(1, 9) = 0.022, p = 0.539] (Figure 5). The results of the probabilistic analysis refer to the param- eter of the probability for overlap of concentric and eccentric frequency distributions at the deﬁned interval in all four angle intervals separately. For angles between 0 and 30◦the median of the probabilities for the overlap of concentric and eccentric fre- quency distributions for all recorded subjects was found to be 69.33%, with a range of 33.04–90.73%. For angles exceeding 30◦ the probability for the overlap of concentric and eccentric fre- quency distributions at the deﬁned interval decreased (Figure 6). The decreased probabilities represent that the frequency distri- butions of concentric and eccentric contractions are more likely to differ from each other. For 30–60◦, 60–90◦, and 90–120◦the FIGURE 4 \| Activation proﬁles of one subject of concentric (blue) and eccentric (pink) contractions of brachioradialis during different elbow ﬂexion and extension angles with the information on the probability of the mean value of sEMG envelope to remain within ±2.5% MVC interval. FIGURE 4 \| Activation proﬁles of one subject of concentric (blue) and eccentric (pink) contractions of brachioradialis during different elbow ﬂexion and extension angles with the information on the probability of the mean value of sEMG envelope to remain within ±2.5% MVC interval. Frontiers in Physiology \| Integrative Physiology Frontiers in Physiology \| Integrative Physiology February 2015 \| Volume 6 \| Article 30 \| 6 Probabilistic analysis of categorized sEMG Von Werder et al. median of the probability for overlap decreased to 5.77% with a range of 0.01–24.98%, 4.88% with a range of 0.02–24.24%, 5.37% with a range of 0.05–28.82%, respectively (Table 1). movement parameters is important when dynamic sEMG record- ings are evaluated. However, to avoid extremely complex mea- suring setups, not all movement parameters must be deﬁned as variable movement factors. DISCUSSION The analysis of sEMG in dynamic contractions contains multiple factors which impact on the analysis of the data and consequently might affect the possible interpretations. Sometimes the analysis of muscular activation during dynamic and complex movements is of high relevance, in particular if pathological movements are to be evaluated. Thereby, less restricted and uncontrollable movement conditions are inevitable and lead to a more complex interpretation of the sEMG. The introduced procedure for the systematic analysis of sEMG recordings during dynamic contraction includes a categorization of the data combined with the analysis of frequency distributions in a probabilistic manner. Independently on a deterministic or probabilistic analysis of dynamic contractions, the ﬁrst step must be assigned to a system- atic categorization of the data. Through the categorization of the data with the help of a decision tree, the possible ﬁndings on mus- cular activation patterns were assigned to a combination of move- ment parameters. The three analyzing scenarios revealed that in the example of ﬂexion and extension of the elbow, the muscular activation of brachioradialis is affected by the variable movement factors contraction type and ﬂexion and extension angle. From the analyzing scenario A one can conclude that concentric con- tractions need a higher normalized sEMG envelope amplitude than eccentric contractions. Additionally, from the analyzing sce- nario B one can conclude that the amplitude of normalized sEMG increases with increasing ﬂexion and extension angle. However, analyzing scenario C highlights that not the assignment of one movement factor is sufﬁcient, but the combination of both move- ment factors is important whenever changes in the muscular activation of brachioradialis are detected. Although analyzing sce- nario A showed for all subjects an impact of contraction type on the muscular activation of brachioradialis, for small angle intervals no difference of the normalized sEMG amplitude for eccentric and concentric contraction were observed. Therefore, the categorization of the data and the assignment to constant FIGURE 5 \| Mean values of sEMG envelope in four different elbow ﬂexion angle intervals with concentric (blue) and eccentric (pink) contractions of brachioradialis. Table 1 \| Overview on the deterministic and probabilistic results of the categorized data of analyzing scenario C. STATISTICAL ANALYSIS But at the same time, the value of the probable overlap in a deﬁned interval accounts for the probability of the negative effect, namely that the concentric and eccentric sEMG do not differ from each other. For angles between 30 and 60◦the median and range of the probability that both frequency distributions in a deﬁned interval do not differ from each other was computed to 5.77% with a range of 0.01–24.98%. Thereby, in four of the 10 subjects the probability exceeded 20%, meaning that for these subjects the tested hypothesis, with respect to the parameter of overlap, holds true with a probability of less than 80%. With the probabilistic parameter additional informa- tion are provided to add a conﬁdence to the obtained results. www.frontiersin.org REFERENCES Anderson, C. V., and Fuglevand, A. J. (2008). Probability-based predic- tion of activity in multiple arm muscles: implications for functional electrical stimulation. J. Neurophysiol. 100, 482–494. doi: 10.1152/jn. 00956.2007 Farina, D. (2006). Interpretation of the surface electromyogram in dynamic con- tractions. Exerc. Sport Sci. Rev. 34, 121–127. doi: 10.1249/00003677-200607000- 00006 Farina, D., Merletti, R., and Enoka, R. M. (2004). The extraction of neural strategies from the surface EMG. J. Appl. Physiol. 96, 1486–1495. doi: 10.1152/japplphys- iol.01070.2003 Greenhouse, S. W., and Geisser, S. (1959). On the methods in the analysis of proﬁle data. Psychometrika 24, 95–112. doi: 10.1007/BF02289823 Guidetti, L., Rivellini, G., and Figura, F. (1996). EMG patterns during running: intra- and inter- individual variability. J. Electromyogr. Kinesiol. 6, 37–48. doi: 10.1016/1050-6411(95)00015-1 Hermens, H., Freriks, B., Disselhorst-Klug, C., and Rau, G. (2000). Development of recommendations for sensors and sensor placement procedures. J. Electromyogr. Kinesiol. 10, 361–374. doi: 10.1016/S1050-6411(00) 00027-4 FIGURE 6 \| The probability that the frequency distributions of concentric and eccentric categorized data overlaps within an interval of 2.5% MVC was computed for four ﬂexion and extension angle intervals. Hug, F. (2011). Can muscle coordination be precisely studied by sur- face electromyography? J. Electromyogr. Kinesiol. 21, 1–12. doi: 10.1016/j.jelekin.2010.08.009 Johnson, L. A., and Fuglevand, A. J. (2009). Evaluation of probabilistic meth- ods to predict muscle activity: implications for neuroprosthetics. J. Neural Eng. 6:055008. doi: 10.1088/1741-2560/6/5/055008 Komi, P. V., Linnamo, V., Silventoinen, P., and Sillanpää, M. (2000). Force and EMG power spectrum during eccentric and concentric actions. Med. Sci. Sports Exerc. 32, 1757–1762. doi: 10.1097/00005768-200010000- 00015 The ranges of the computed probabilities for the overlap of both frequency distributions reveal that there are inter-individual dif- ferences for the activation of brachioradialis in concentric and eccentric contractions along all angle areas. Linnamo, V., Strojnik, V., and Komi, P. V. (2006). Maximal force during eccentric and isometric actions at different elbow angles. Eur. J. Appl. Physiol. 96, 672–678. doi: 10.1007/s00421-005-0129-x For the deterministic analysis the outcome of the statistical test is to reject or hold the raised hypothesis. In contrast, if the analysis is connected to the test of a speciﬁc hypothesis with a probabilistic approach, additional information on the likelihood that the hypothesis holds true or fails are available which adds a confidence to the results. More probabilistic methods could be added to the evaluation of two frequency distributions. REFERENCES p J y p y 2015. Citation: Von Werder SCFA, Kleiber T and Disselhorst-Klug C (2015) A method for a categorized and probabilistic analysis of the surface electromyogram in dynamic contractions. Front. Physiol. 6:30. doi: 10.3389/fphys.2015.00030 This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology. Copyright © 2015 Von Werder, Kleiber and Disselhorst-Klug. 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 permit- ted, 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. Citation: Von Werder SCFA, Kleiber T and Disselhorst-Klug C (2015) A method for a categorized and probabilistic analysis of the surface electromyogram in dynamic contractions. Front. Physiol. 6:30. doi: 10.3389/fphys.2015.00030 This article was submitted to Integrative Physiology, a section of the journal Frontiers in Physiology. Copyright © 2015 Von Werder, Kleiber and Disselhorst-Klug. 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 permit- ted, 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. Copyright © 2015 Von Werder, Kleiber and Disselhorst-Klug. 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 permit- ted, 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. REFERENCES Besides the similarity or difference of two frequency distributions one could also assess the probability that one sEMG distribution possess higher sEMG amplitudes than the other. Nakazawa, K., Kawakami, Y., Fukunaga, T., Yano, H., and Miyashita, M. (1993). Differences in activation patterns in elbow ﬂexor muscles during isometric, concentric and eccentric contractions. Eur. J. Appl. Physiol. Occup. Physiol. 66, 214–220. doi: 10.1007/BF00235096 Perry, J. (1992). Gait Analysis: Normal and Pathological Function. Thorofare, NJ: SLACK Incorporated. Rau, G., Disselhorst-Klug, C., and Schmidt, R. (2000). Movement biomechan- ics goes upwards: from the leg to the arm. J. Biomech. 33, 1207–1216. doi: 10.1016/S0021-9290(00)00062-2 Rau, G., Schulte, E., and Disselhorst-Klug, C. (2004). From cell to movement: to what answers does EMG really contribute? J. Electromyogr. Kinesiol. 14, 611–617. doi: 10.1016/j.jelekin.2004.02.001 The main potential of the probabilistic approach lies in the fact that each possible value of the normalized sEMG can be assigned to a probability that this value will occur in a speciﬁc interval. In combination with parameters, which describe the similarity or difference of two frequency distributions, the information on the likelihood that the analyzed effect occurs or not occurs can be provided. It can be assumed, that inter-individual differ- ences increase with more uncontrollable movement conditions in free movements. A measure on the conﬁdence of the detected results could account for this increasing effect of inter-individual differences. Schmidt, R., Disselhorst-Klug, C., Silny, J., and Rau, G. (1999). A marker-based measurement procedure for unconstrained wristand elbow motions. J. Biomech. 32, 615–621. doi: 10.1016/S0021-9290(99)00036-6 Williams, S., Schmidt, R., Disselhorst-Klug, C., and Rau, G. (2006). An upper body model for the kinematical analysis of the joint chain of the human arm. J. Biomech. 39, 2419–2429. doi: 10.1016/j.jbiomech.2005.07.023 Conﬂict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Received: 31 October 2014; accepted: 19 January 2015; published online: 11 February 2015. Received: 31 October 2014; accepted: 19 January 2015; published online: 11 February 2015. Analyzing sEMG signals during dynamic contractions is chal- lenging. However, the introduced procedure of analyzing cate- gorized data with a probabilistic approach allows to address the challenge of the inﬂuence of different movement conditions on the muscular activation and provides additional information on the conﬁdence of the results by adding probabilities to the tested hypothesis. DISCUSSION Angle Deterministic approach Probabilistic approach interval [◦] Two Way repeated ANOVA probability overlap [%] F(1,9) p-value Median Range 0–30 0.022 0.539 69.33 33.04–90.73 30–60 21.743 0.002 5.77 0.01–24.98 60–90 27.803 0.001 4.88 0.02–24.24 90–120 36.070 <0.0005 5.37 0.05–28.82 Table 1 \| Overview on the deterministic and probabilistic results of the categorized data of analyzing scenario C. FIGURE 5 \| Mean values of sEMG envelope in four different elbow ﬂexion angle intervals with concentric (blue) and eccentric (pink) contractions of brachioradialis. February 2015 \| Volume 6 \| Article 30 \| 7 Probabilistic analysis of categorized sEMG Von Werder et al. FIGURE 6 \| The probability that the frequency distributions of concentric and eccentric categorized data overlaps within an interval of 2.5% MVC was computed for four ﬂexion and extension angle intervals. ACKNOWLEDGMENT This work was in part funded by the German Research School for Simulation Sciences, Jülich, Germany. February 2015 \| Volume 6 \| Article 30 \| 8 Frontiers in Physiology \| Integrative Physiology
https://openalex.org/W2524680273	https://europepmc.org/articles/pmc5059534?pdf=render	English	null	Dietary Chitosan Supplementation Increases Microbial Diversity and Attenuates the Severity of<i>Citrobacter rodentium</i>Infection in Mice	Mediators of inflammation	2,016	cc-by	5,736	Guiping Guan,1,2 Hongbing Wang,3 Shuai Chen,2 Gang Liu,2 Xia Xiong,2 Bie T Veeramuthu Duraipandiyan,4 Naif Abdullah Al-Dhabi,4 and Jun Fang1 Guiping Guan,1,2 Hongbing Wang,3 Shuai Chen,2 Gang Liu,2 Xia Xiong,2 Bie Tan,2 Veeramuthu Duraipandiyan,4 Naif Abdullah Al-Dhabi,4 and Jun Fang1 1College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, China 2Key Laboratory of Agroecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Hunan Co-Innovation Center of Animal Production Safety, Hunan 410125, China 3Hunan Institute of Animal and Veterinary Science, Changsha, Changsha 410131, China 4Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P.O. Box. 2455, Riyadh 11451, Saudi Arabia 1College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, China 2Key Laboratory of Agroecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences, Hunan Provincial Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Hunan Co-Innovation Center of Animal Production Safety, Hunan 410125, China 3Hunan Institute of Animal and Veterinary Science, Changsha, Changsha 410131, China 4Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P.O. Box. 2455, Riyadh 11451, Saudi Arabia Correspondence should be addressed to Gang Liu; gangle.liu@gmail.com Correspondence should be addressed to Gang Liu; gangle.liu@gmail.com Received 2 June 2016; Revised 25 August 2016; Accepted 14 September 2016 Received 2 June 2016; Revised 25 August 2016; Accepted 14 September 2016 Academic Editor: Jos´e C´esar Rosa Neto Academic Editor: Jos´e C´esar Rosa Neto Copyright © 2016 Guiping Guan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. C57BL/6 mice were tested in order to investigate the effects of dietary chitosan (COS) supplements on intestinal microflora and resistance to Citrobacter rodentium infection. The findings reveal that, after consuming a 300 mg/kg COS diet for 14 days, microflora became more diverse as a result of the supplement. Mice receiving COS exhibited an increase in the percentage of Bacteroidetes phylum and a decrease in the percentage of Firmicutes phylum. After Citrobacter rodentium infection, the histopathology scores indicated that COS feeding resulted in less severe colitis. Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2016, Article ID 9236196, 7 pages http://dx.doi.org/10.1155/2016/9236196 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2016, Article ID 9236196, 7 pages http://dx.doi.org/10.1155/2016/9236196 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2016, Article ID 9236196, 7 pages http://dx.doi.org/10.1155/2016/9236196 Hindawi Publishing Corporation Mediators of Inﬂammation Volume 2016, Article ID 9236196, 7 pages http://dx.doi.org/10.1155/2016/9236196 Guiping Guan,1,2 Hongbing Wang,3 Shuai Chen,2 Gang Liu,2 Xia Xiong,2 Bie T Veeramuthu Duraipandiyan,4 Naif Abdullah Al-Dhabi,4 and Jun Fang1 IL-6 and TNF-𝛼were significantly lower in colon from COS-feeding mice than those in the control group. Furthermore, mice in COS group were also found to experience inhibited activation of nuclear factor-kappa B (NF-𝜅B) in the colonic tissue. Overall, the findings revealed that adding 300 mg/kg COS to the diet changed the composition of the intestinal microflora of mice, resulting in suppressed NF-𝜅B activation and less production of TNF-𝛼and IL-6; and these changes led to better control of inflammation and resolution of infection with C. rodentium. 1. Introduction Until being ready for analysis, samples were kept at −80∘C. The human intestinal tract houses 500–1,000 species of microbiota at a total quantity of almost 100 trillion [11]. Various factors, including location and diet, have a significant impact on the metagenome, although these factors do not have the same effect on a single organism’s genome. Pathogen displacement, the extraction of energy such as SCFAs from nondigestible dietary substrates, and the development of the immune system all depend on intestinal microbiota. Significant changes in the natural microbiota homeostasis have been linked to various illnesses in humans [12]. 2.2. C. rodentium Infection and Monitoring. After giving either the basal or COS diet for a period of 14 days, 10 mice in each group were infected with C. rodentium. CO2 asphyxiation was administered to all mice on D7 after infection. Samples of the mice’s feces, tissue, and colonic content were gathered. Bacteria were grown in Luria Bertani broth (0.05 g/L nalidixic acid/mL) overnight. Centrifugation was used on the cultures and the pellet produced was resuspended in sterile phosphate-buffered saline (PBS). This resulted in a concentration of 5 × 109 CFU/mL. Mice were then challenged at 9 am and 5 × 109 CFU C. rodentium were orally administered to the mice using the gavage process. In order to prevent shedding mice from spreading infection, all mice were kept separately when infected with C. rodentium. The mice were fed with the same basal or COS diet for 7 days, respectively. Feces collection was conducted on D7 after infection, at which point they were also weighed and suspended in PBS. Serial dilutions were plated onto plates containing nalidixic acid. After a period of 24 hours, bacterial colonies were counted. Each dilution was grown at 37∘C overnight using the pour plate method in MacConkey agar supplemented with the antibiotic kanamycin (40 𝜇g/mL) in order to compute only C. rodentium. COS is among the most plentiful polysaccharides found in insects, fungi, squid, oysters, krill, clams, and shellfish. It is a natural N-deacetylated derivative of chitin [13]. Many researchers have explored the ways in which the expression of Th1 and Th2 cytokines is impacted by COS [14]. In pigs [14], mice [15], rats [16], and fishes [17], COS serves as a regulator of the immune response. 1. Introduction infiltration, are prompted by A/E pathogens following the colonization of the colonic epithelium [4]. C. rodentium amongst mice populations has been used by many researchers to represent intestinal E. coli since intestinal EPEC or EHEC does not infect mice. Citrobacter rodentium, a mucosal pathogen found in mice, and enteropathogenic Escherichia coli (EPEC) and entero- haemorrhagic E. coli (EHEC), enteric pathogens found in humans, create attaching and effacing (A/E) lesions that result in the colonization of mucosa within the intestinal tract [1, 2]. The intestinal epithelium becomes infiltrated by bacterial attachments, the brush border microvilli become depleted, and pedestal-shaped structures begin to form beneath the adherent bacterium [3]. Colonic tissue changes comparable to EHEC and EPEC infection, along with a rise in the production of inflammatory cytokine and leukocyte A number of significant intestinal disorders found in humans are also frequently modelled by C. rodentium in mice, such as colon tumorigenesis, ulcerative colitis, and Crohn’s disease [5]. Colitis develops in mice that have been infected with C. rodentium, which results in the bacteria becoming overabundant and the mice’s natural microbiota reducing in variety and quantity [6]. When infected, 1–3% of the mice’s intestinal microbiota becomes C. rodentium [7], whilst the 2 Mediators of Inflammation colon comes under attack by 109 colony forming units (CFUs) per g [8]. The genetic makeup of the mouse impacts the formation of C. rodentium-induced colitis, with mice such as C3H/HeJ and FVB/N being prone to develop colitis as a result of the infection and mice such as CD-1 and C57BL/6 being considered amongst those that are not prone to develop colitis and are only prone to subclinical symptoms [9]. Vulnerability to contracting C. rodentium, as well as tendency to show a certain immune response, has been found to be highly related to intestinal microbiota composition [10].h to the COS group, which was consumed for 14 consecutive days. COS has a 6-sugar unit of N-acetyl glucosamine with 𝛽-(1–4)-linkages [8], an average molecular weight of less than 1,000 Da, and a degree of deacetylation over 95% and is free of endotoxins. The COS provided to the mice in this study was provided by the Chinese Academy of Sciences’ Dalian Chemical and Physical Institute. The findings of an earlier research paper [14] were used as a guideline for the duration and amount of COS given. Throughout the experiment, the mice’s weight gain, water consumption, and food consumption were recorded. 1. Introduction However, little is known about how intestinal microbiota is impacted by dietary COS despite a small number of studies attempting to study this in pigs and chickens [18]. It would be useful to gain insight into whether it is via the intestinal microbiota that COS is able to perform its advantageous biological functions, since it is already known that a number of biological functions are impacted by the intestinal microbiota [19, 20]. Nonetheless, at present, few findings exist on the topic of C. rodentium, intestinal micro- biota, and the effects of COS supplements on them. Thus, the objective of this study is to evaluate the effects of COS sup- plementation on microbial flora composition changes, proin- flammatory molecule reduction (and/or anti-inflammatory molecule increase), and C. rodentium infection. 2.3. Colon Tissue: Histological Analysis. The colon of each mouse was removed and fixed in 10% formalin. Hematoxylin and eosin were used to stain and prepare paraffin-embedded sections. Six criteria (edema, ulcers, erosion, inflammation, goblet cell hyperplasia, and cryptitis) were outlined in order to histologically grade the colitis. A scale of 0–4 was used to score the lesions as follows: no epithelial thickening/colitis (0); minor epithelial cell hyperplasia/greater quantity of mucosa leukocytes (1); inflammation at numerous loci, sub- mucosa, and mucosa infiltrated by leukocytes and/or signifi- cant epithelial cell hyperplasia (with 2-3 times’ rise in crypts) (2); greater epithelial cell hyperplasia (3–10 times higher crypt count), reduction of goblet cells secreting mucin, ulceration, and/or significant leukocytic infiltration of the submucosa and mucosa (3); extremely high epithelial cell hyperplasia (10 or more times higher crypt count), crypt abscesses, and/or serious infiltration of transmural leukocytes (4). 2. Materials and Methods Biotree, a commercial firm based in Shanghai, carried out the general data analyses and Illumina MiSeq sequencing. Previous findings were used to guide the MiSeq PE Libraries, MiSeq sequencing, and additional analysis [22]. The corresponding authors can be contacted to request further information on references, raw data, and the sequencing run. period. Prior to conducting the analysis with Alpha Imager 2200 (Alpha Innotech Corporation, CA, USA), overnight incubation at 4∘C was carried out with the primary antibod- ies, whilst 60-minute incubation at room temperature was carried out with the HRP-conjugated secondary antibodies. Finally, electronic quantification and normalization of signal intensity to Lamin B protein abundance were performed. 2.8. Statistical Data Analysis. SPSS 22.0 (Chicago, IL, USA) was used to carry out all statistical analysis. The data illus- trated in this section are the means ± the standard error of the mean (SEM). Student’s 𝑡-test was used to analyze the data between two groups. In this study, statistical significance is shown in values of 𝑃< 0.05. 2.8. Statistical Data Analysis. SPSS 22.0 (Chicago, IL, USA) was used to carry out all statistical analysis. The data illus- trated in this section are the means ± the standard error of the mean (SEM). Student’s 𝑡-test was used to analyze the data between two groups. In this study, statistical significance is shown in values of 𝑃< 0.05. 2. Materials and Methods 2.1. Mice and Diet. This study used male C57BL/6 mice aged 6–8 weeks old, bred and reared at Hunan Agricultural University by breeders from China’s Changsha-based SLAC Laboratory Animal Center. The reason for using only male mice was that sex and maternal factors have been shown to impact microflora composition. Hunan Agricultural Univer- sity’s Animal Care and Use Committees provided approval for the experiments. The mice were provided plentiful water and a normal diet [21], and they were kept individually in animal colonies that were free from pathogens. The colonies were kept at a temperature of 25∘C, relative humidity of 53%, and an equal balance of 12 hours of dark/light. The mice were assigned at random to the COS and control groups after being housed in the colonies for three days, with 20 mice in each group. A normal rodent diet [21] was given to the control group. 300 mg/kg COS was added to the standard diet given 2.4. 16S rDNA and Illumina MiSeq Sequencing. After giving either the basal or COS diet for a period of 14 days, feces were collected and 10 mice in each group were killed to collect colon contents. Then, feces and colon contents were used 3 Mediators of Inflammation Mediators of Inflammation Table 1: Comparative results of 16S rRNA gene libraries’ phylotype coverage and diversity at 97% similarity based on pyrosequencing analysis (𝑛= 6). Number of reads Number of OUT Coverage Richness estimator Diversity index Ace (95% Cl) Chao (95% Cl) Shannon (95% Cl) Simpson (95% Cl) Colon Control 12,568 37 99.88% 53 (43–86) 58 (43–112) 0.72 (0.69–0.74) 0.68 (0.66–0.70) COS 12,794 46 99.88% 62 (52–87) 56 (50–81) 0.88 (0.85–0.90) 0.59 (0.58–0.60) Feces Control 11,521 314 99.90% 324 (312–344) 329 (314–357) 4.17 (4.23–4.39) 0.025 (0.024–0.026) COS 10,956 271 99.43% 304 (289–333) 302 (281–337) 4.05 (4.01–4.08) 0.033 (0.032–0.034) for 16S rDNA sequencing. As per the guidelines for DNA isolation, the QiagenQIAamp DNA Stool Mini Kit was used to extract DNA from luminal colon and feces contents. In order to create a baseline sample for each sample type, equal quantities of DNA were gathered from six individual mice. Primers 515F 5󸀠-GTGCCAGCMGCCGCGG-3󸀠and 907R 5󸀠- CCGTCAATTCMTTTRAGTTT-3󸀠(the barcode being an eight-base sequence individual to each specific sample) were used to amplify the V4-V5 region of the bacteria 16S riboso- mal RNA gene by PCR. 3. Results 2.5. Analysis of Colonic mRNA. IL-6 and TNF-𝛼expression were incorporated in the analyses, which occurred after the collection and weighing of the proximal colon. TRIZOL regent (Invitrogen, USA) was used for the extraction of mRNA. Reverse transcription of the cDNA was conducted, and an ABI 7500 Fast RT-PCR machine (Applied Biosys- tems), Superscript II reverse transcriptase (Invitrogen), and oligo (dT) 20 were used to carry out real-time PCR. According to the weight measurements, there was no postin- fection change in the weight of each mouse in either of the two groups. Additionally, as shown in Figure 1, at D7 after infection, no change was noted in the quantity of C. rodentium in the feces or colon contents of each group. However, as shown in Figure 2, a significant increase in colitis severity was observed at D7 after infection amongst the control group of mice compared to the mice provided with COS. 2.6. Cytokine Measurement. A 50 mM Tris-HCl with 10 𝜇g/ mL protease inhibitor solution (Sigma-Aldrich Co., USA) was used to homogenise the colonic samples over ice. Cen- trifugation of 20 minutes was used on the homogenates at 30,000 ×g (4∘C). Following centrifugation, a Sandwich ELISA Kit (ELISA Ready-SET-GO, eBioscience, CA, USA) was used to test the supernatants for TNF-𝛼and IL-6. Normalization of cytokine was carried out to match the colonic samples’ protein levels. As shown in Table 1, 16S rDNA sequencing was used to analyze the intestinal microbiota once the experiment was completed. The results of 16S rDNA sequencing were used to investigate the effects of COS supplements on histopathology scores. It is clear that the COS-fed mice showed reduced microbiota diversity (as per their feces samples), according to the Shannon and Simpson indices, compared to the control group of mice. The indices also showed that the COS-fed mice showed higher microbiota diversity than the control group of mice based on the colon analysis. Interestingly, it was indicated by the Ace and Chao richness indices that both groups of mice showed similar microbiota community richness in their faecal and colonic samples (Table 1).hi 2.7. NF-𝜅B (p65) Immunoblotting. The NF-𝜅B (p65) tran- scription factor assay kit (Cayman Chemical Company, MI, USA) was used to measure NF-𝜅B (p65) binding activity in the nuclear extracts. 3. Results The proteins extracted from nuclear or cytoplasmic fractions were taken in equal quantities and then (1) divided using SDS-PAGE, (2) relocated to PVDF membranes (Millipore, MA, USA), and obstructed using 5% nonfat milk in a Tris-Tween buffered saline buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) over a 3-hour 2.7. NF-𝜅B (p65) Immunoblotting. The NF-𝜅B (p65) tran- scription factor assay kit (Cayman Chemical Company, MI, USA) was used to measure NF-𝜅B (p65) binding activity in the nuclear extracts. The proteins extracted from nuclear or cytoplasmic fractions were taken in equal quantities and then (1) divided using SDS-PAGE, (2) relocated to PVDF membranes (Millipore, MA, USA), and obstructed using 5% nonfat milk in a Tris-Tween buffered saline buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20) over a 3-hour The RDP classifier was used to perform a taxon- dependent analysis in order to identify the intestinal micro- biota’s taxonomy. For both groups of mice, the faecal micro- biota showed nine phyla, including one candidate division (TM7), whilst the colon microbiota showed seven phyla, including one candidate division (TM7). Additionally, six 4 Mediators of Inflammation 10 8 6 4 2 0 Log CFU/g COS Control COS Control Feces Colon contents Figure 1: D7 postinfection C. rodentium levels in colon contents and feces of infected C57BL/6 mice. Feces and colon contents were gathered before undergoing homogenisation and being plated in serial dilution on LB agar (𝑛= 10). 10 8 6 4 2 0 Log CFU/g COS Control COS Control Feces Colon contents Figure 1: D7 postinfection C. rodentium levels in colon contents d f f i f t d C57BL/6 i F d l t t Firmicutes Bacteroidetes Proteobacteria Others 0 20 40 60 80 100 Phylum percentage (%) COS Control COS Control Feces Colon contents Figure 3: D7 postinfection composition of the intestinal microbiota Microbial composition in the colon and feces of both groups (𝑛= 6) Control mice were given standard drinking water and a basal rodent diet, whilst COS mice were given the same water and diet with the addition of COS at 300 mg/kg. Firmicutes 0 20 40 60 80 100 Phylum percentage (%) COS Control Colon contents COS ces Control Figure 1: D7 postinfection C. rodentium levels in colon contents and feces of infected C57BL/6 mice. 3. Results Feces and colon contents were gathered before undergoing homogenisation and being plated in serial dilution on LB agar (𝑛= 10). Figure 3: D7 postinfection composition of the intestinal microbiota. Figure 3: D7 postinfection composition of the intestinal microbiota. Microbial composition in the colon and feces of both groups (𝑛= 6). Control mice were given standard drinking water and a basal rodent diet, whilst COS mice were given the same water and diet with the addition of COS at 300 mg/kg. Figure 3: D7 postinfection composition of the intestinal microbiota. Microbial composition in the colon and feces of both groups (𝑛= 6). Control mice were given standard drinking water and a basal rodent diet, whilst COS mice were given the same water and diet with the addition of COS at 300 mg/kg. D7 after infection Control COS B A 0 1 2 3 4 Histopathology scores Figure 2: D7 postinfection histopathology scores of each mouse (𝑛= 10). Histopathology scores The purpose of this was to identify whether the beneficial outcomes of COS (i.e., in reducing infection) are supported by the suppression of NF-𝜅B activation pathways. The results showed that the control group of mice had significantly higher nuclear NF-𝜅B (p65) in their colonic samples than the COS-fed group of mice, which suggests that COS suppresses NF-𝜅B activation (see Figure 5). 4. Discussion Mediators of Inflammation 5 Mediators of Inflammation TNF-𝛼 IL-6 B B A A 0 1 2 3 4 Relative mRNA abundance Control COS (a) Cytokine concentration TNF-𝛼 IL-6 B B A A 0 200 400 600 800 (pg/mg protein) Control COS (b) Figure 4: Control and COS group (𝑛= 6) mucosal inflammatory responses. (a) RT-PRC evaluation of IL-6 and TNF-𝛼mRNA level. (b) ELISA evaluation of IL-6 and TNF-𝛼protein level. Cytokine concentration TNF-𝛼 IL-6 B B A A 0 200 400 600 800 (pg/mg protein) Control COS (b) TNF-𝛼 IL-6 B B A A 0 1 2 3 4 Relative mRNA abundance Control COS ( ) (b) (a) Figure 4: Control and COS group (𝑛= 6) mucosal inflammatory responses. (a) RT-PRC evaluation of IL-6 and TNF-𝛼mRNA level. (b) ELISA evaluation of IL-6 and TNF-𝛼protein level. B A COS Control p-p65 Lamin B Nuclear phosphorylated p65 (p-p65) 0.0 0.5 1.0 1.5 2.0 Relative protein abundance (b) p65 Lamin B Nuclear NF-𝜅B (p65) B A COS Control 0.0 0.2 0.4 0.6 0.8 1.0 Relative protein abundance (a) p65 Lamin B Nuclear NF-𝜅B (p65) B A COS Control 0.0 0.2 0.4 0.6 0.8 1.0 Relative protein abundance (a) B A COS Control p-p65 Lamin B Nuclear phosphorylated p65 (p-p65) 0.0 0.5 1.0 1.5 2.0 Relative protein abundance (b) Figure 5: Immunoblotting of (a) nuclear NF-𝜅B (p65) and (b) phosphorylated nuclear NF-𝜅B (p65) in control and COS group (𝑛= 6). (a) (b) Figure 5: Immunoblotting of (a) nuclear NF-𝜅B (p65) and (b) phosphorylated nuclear NF-𝜅B (p65) in control and COS group (𝑛= 6). The results noted no impact on the quantity of C. rodentium taken from faecal samples, which suggests that the regulating impact of COS is not due to swift pathogenic eradication but rather due to an improvement in microflora diversity. Various biological functions are said to be influenced by such microbiota [19, 20], and it has been associated with cancer [24, 25], cirrhosis of the liver [26], and other diseases. Since Firmicutes has the ability to offer extra energy to the host by fermenting plant polysaccharide to SCFA, this is the reason for obesity being linked to higher concentrations of Firmicutes: Bacteroidetes [27]. At present, the reason behind COS’s minimising impact on Firmicutes quantities is unclear. 4. Discussion C. rodentium, which is similar to the human enteropath- ogenic E. coli infection, is an extracellular enteric pathogen that infects mice. It has also been noted that inflammatory bowel disease (IBD) is often modelled using C. rodentium in mice. IBD results in intestinal inflammation. Thus, intestinal pathology is regulated by inflammatory regulators such as inducible TNF𝛼and IL-6 [6]. Intestinal bacterial communi- ties influence mice’s susceptibility and ability to overcome the C. rodentium infection, as does the immune response of the mice [23]. E. coli is commonly modelled by the infection of mice with C. rodentium since mice are not affected by EPEC or EHEC. C. rodentium has been found in all mice strains but it is rare in human disease. Depending on the strain, an infected mouse can suffer subclinical disease or death after infection [9]. For instance, strains believed to be resistant to developing colitis as a result of C. rodentium infection include CD-1 and C57BL/6 mice. On the other hand, it is believed that susceptibility is present amongst mice strains C3H/HeJ and FVB/N [9]. Based on this information, human E. coli infection was modelled in this study by infecting C57BL/6 mice with C. rodentium.ht Figure 2: D7 postinfection histopathology scores of each mouse (𝑛= 10). phyla were found in each of the mice groups. As illustrated in Figure 3, in colon contents, Firmicutes (96.8%) and Pro- teobacteria (1.3%) were the phyla with the highest percentages in the colon microbiota of the COS-fed mice, whilst in feces Firmicutes (96.1%) and Proteobacteria (1.8%) were the phyla with the highest percentages in the control mice. The percentages of Bacteroidetes (61.4%), Firmicutes (27.1%), and Proteobacteria (4.5%) were the three most abundant in the faecal microbiota of the COS-fed mice, whilst the percentages of Bacteroidetes (53.8%), Firmicutes (39.6%), and Proteobacteria (3.5%) were the most abundant in the control mice. As shown in Figure 4(a), colon analysis of control mice showed significantly higher IL-6 and TNF-𝛼mRNA than the COS-fed mice at D7 after infection. This finding is supported through the ELISA results (Figure 4(b)). The results showed that, after infecting the mice with C. rodentium, inflammation was resolved more quickly amongst mice receiving 300 mg/kg COS than the control mice. Nuclear NF-𝜅B (p65) measurements were taken to iden- tify the activation of NF-𝜅B in the colons of the mice. References occurs is related to microflora changes and not inflammation [29]. The results of this study indicate that the effects of COS on the intestines only occur in specific areas of the gut and only take on a specific form. Mice fed with COS were found to have a lower expression of inflammatory cytokines, which is linked to bacterial flora shifts that lead to faster infection recovery. [1] D. B. Schauer and S. Falkow, “Attaching and effacing locus of a Citrobacter freundii biotype that causes transmissible murine colonic hyperplasia,” Infection and Immunity, vol. 61, no. 6, pp. 2486–2492, 1993. [2] S. A. Luperchio, J. V. Newman, C. A. Dangler et al., “Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C. rodentium and mouse-pathogenic Escherichia coli,” Journal of Clinical Microbiology, vol. 38, no. 12, pp. 4343–4350, 2000. y COS is a d-glucosamine oligomer that cannot be eroded by enzymes in the gut. Thus, COS directly protects the host’s intestinal epithelial cells (IEC). Research indicates that IEC expresses various chemokine and cytokine receptors such as TLR4 and other Toll-like receptors [30]. In the IEC, NF-𝜅B-regulated proinflammatory cytokine production is promoted by the interaction between TNF-𝛼and their respective receptors, TLR4, TNFR1, and TNFR2 [31]. Studies also show that dysfunction of the intestinal epithelial barrier can occur as a result of pronounced IEC-regulated mucosal inflammatory responses, which are associated with greater TLR4 and TNFR expression [32]. In the current study, it was revealed that a significant reduction in the activation of NF-𝜅B along with colon production of TNF-𝛼and IL-6 was achieved through the administration of COS through the basal diet. Since recent studies have found that mice can develop disease sharing similar characteristics to Crohn’s as a result of IEC-derived TNF-𝛼[32], this indicates that the anti-inflammatory impact of COS could be the result of COS’ suppressive impact on the activation of NF-𝜅B and production of proinflammatory cytokines in the IEC. [3] G. Frankel and A. D. Phillips, “Attaching effacing Escherichia coli and paradigms of Tir-triggered actin polymerization: get- ting off the pedestal,” Cellular Microbiology, vol. 10, no. 3, pp. 549–556, 2008. [4] T. K. Lapointe, P. M. O’Connor, and A. G. Buret, “The role of epithelial malfunction in the pathogenesis of enteropathogenic E. coli-induced diarrhea,” Laboratory Investigation, vol. 89, no. 9, pp. 964–970, 2009. [5] P. Chandrakesan, B. Roy, L. U. M. R. 4. Discussion Some studies have also shown that a reduction in intestinal Firmicutes can be achieved through acidic oligosaccha- rides, a galactooligosaccharides/long-chain fructan solution (GOS/lcF, 9/1), fructooligosaccharides, and other oligosac- charides [28]. It may be that the N-acetyl glucosamine of COS may function as a binding agent that allows COS to influence the attachment of bacteria to the intestine. Given this, research on pigs and chickens indicates that intestinal microbial communities can be impacted by the introduction of COS supplements [18]. Furthermore, intestinal microbiota may change as a result of COS’s role as a fermentable substrate for certain bacteria, as this could lower the pH level of the gut and activate natural acid production [26]. Various studies have indicated in vitro and in vivo anti- inflammatory effects of COS. In the current study, it has been found that IL-6 and TNF-𝛼expression in the colon are reduced by COS, whilst microbial flora is impacted by the inflammation [23]. Thus, microbiome changes may be influenced by increased inflammation. Since the faecal sam- ples showed no relationship between C. rodentium quantities and the introduction of COS, this finding is contradictory. Consequently, the speed at which C. rodentium resolution Mediators of Inflammation 6 References Jakkula et al., “Utility of a bacterial infection model to study epithelial-mesenchymal transition, mesenchymal-epithelial transition or tumorigene- sis,” Oncogene, vol. 33, no. 20, pp. 2639–2654, 2014. [6] R. Mundy, T. T. MacDonald, G. Dougan, G. Frankel, and S. Wiles, “Citrobacter rodentium of mice and man,” Cellular Microbiology, vol. 7, no. 12, pp. 1697–1706, 2005. [7] C. Lupp, M. L. Robertson, M. E. 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Kanai et al., “Signaling pathway via TNF-𝛼/NF-𝜅B in intestinal epithelial cells may be directly
https://openalex.org/W2946000217	https://europepmc.org/articles/pmc6718169?pdf=render	English	null	Hypothesis-generating and confirmatory studies, Bonferroni correction, and pre-specification of trial endpoints	Acta orthopaedica	2,019	cc-by	825	Hypothesis-generating and confirmatory studies, Bonferroni correction, and pre-specification of trial endpoints ical Journal Editors) and the WHO also require registration of clinical trials and their endpoints in a publicly accessible register before enrollment of the first participant. Changing endpoints in a randomized trial after its initiation can in some cases be acceptable, but this is never a trivial problem (Evans 2007) and must always be described to the reader. Many authors do not understand the importance of pre-specification and desist from registering their trial, use vague or ambigu- ous endpoint definitions, redefine the primary endpoint during the analysis, switch primary and secondary outcomes, or present completely new endpoints without mentioning this to the reader. Such publications are simply not credible, but are nevertheless surprisingly common (Ramagopalan et al. 2014) even in high impact factor journals (Goldacre et al. 2019). A serious editorial evaluation of manuscripts presenting confir- matory results should always include a verification of the end- point’s pre-specification. A p-value presents the outcome of a statistically tested null hypothesis. It indicates how incompatible observed data are with a statistical model defined by a null hypothesis. This hypothesis can, for example, be that 2 parameters have identi- cal values, or that they differ by a specified amount. A low p-value shows that it is unlikely (a high p-value that it is not unlikely) that the observed data are consistent with the null hypothesis. Many null hypotheses are tested in order to gen- erate study hypotheses for further research, others to con- firm an already established study hypothesis. The difference between generating and confirming a hypothesis is crucial for the interpretation of the results. Presenting an outcome from a hypothesis-generating study as if it had been produced in a confirmatory study is misleading and represents methodologi- cal ignorance or scientific misconduct. Hypothesis-generating studies differ methodologically from confirmatory studies. A generated hypothesis must be confirmed in a new study. An experiment is usually required for confirmation as an observational study cannot provide unequivocal results. For example, selection and confounding bias can be prevented by randomization and blinding in a clin- ical trial, but not in an observational study. Confirmatory stud- ies, but not hypothesis-generating studies, also require control of the inflation in the false-positive error risk that is caused by testing multiple null hypotheses. The phenomenon is known as a multiplicity or mass-significance effect. © 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of the Nordic Orthopedic Federation. 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. DOI 10 1080/17453674 2019 1612624 Acta Orthopaedica 2019; 90 (4): 297 297 Hypothesis-generating and confirmatory studies, Bonferroni correction, and pre-specification of trial endpoints A method for correcting the significance level for the multiplicity effect has been devised by the Italian mathematician Carlo Emilio Bonferroni. The correction (Bender and Lange 2001) is often misused in hypothesis-generating studies, often ignored when designing confirmatory studies (which results in underpow- ered studies), and often inadequately used in laboratory stud- ies, for example when an investigator corrects the significance level for comparing 3 experimental groups by lowering it to 0.05/3 = 0. 017 and believes that this solves the problem of testing 50 null hypotheses, which would have required a cor- rected significance level of 0.05/50 = 0.001. Hypothesis-generating studies are much more common than confirmatory, because the latter are logistically more complex, more laborious, more time-consuming, more expensive, and require more methodological expertise. However, the result of a hypothesis-generating study is just a hypothesis. A hypothe- sis cannot be generated and confirmed in the same study, and it cannot be confirmed with a new hypothesis-generating study. Confirmatory studies are essential for scientific progress. Bender R, Lange S. Adjusting for multiple testing: when and how? J Clin Epidemiol 2001; 54: 343-9. Jonas Ranstam, Statistical Editor jonas.ranstam@gmail.com Jonas Ranstam, Statistical Editor jonas.ranstam@gmail.com Bender R, Lange S. Adjusting for multiple testing: when and how? J Clin Epidemiol 2001; 54: 343-9. Evans S. When and how can endpoints be changed after initiation of a ran- domized clinical trial? PLoS Clin Trials 2007; 2: e18. Goldacre B, Drysdale H, Milosevic I, Slade E, Hartley P, Marston C, Powell- Smith A, Heneghan C, Mahtani K R. COMPare: a prospective cohort study correcting and monitoring 58 misreported trials in real time. Trials 2019; 20: 118. In a confirmatory study, it is mandatory to show that the tested hypothesis has been pre-specified. A study protocol or statistical analysis plan should therefore be enclosed with the study report when submitted to a scientific journal for publica- tion. Since 2005 the ICMJE (International Committee of Med- Ramagopalan S, Skingsley A P, Handunnetthi L, Klingel M, Magnus D, Pak- poor J, Goldacre B. Prevalence of primary outcome changes in clinical trials registered on ClinicalTrials.gov: a cross-sectional study. F1000Re- search 2014, 3: 77.
https://openalex.org/W2855701802	https://bmcnephrol.biomedcentral.com/track/pdf/10.1186/s12882-018-0977-3	English	null	NGAL protects against endotoxin-induced renal tubular cell damage by suppressing apoptosis	BMC nephrology	2,018	cc-by	7,652	Open Access Mei Han1, Ying Li2, Di Wen3, Maodong Liu2, Yuteng Ma1 and Bin Cong3 Mei Han1, Ying Li2, Di Wen3, Maodong Liu2, Yuteng Ma1 and Bin Cong3 Mei Han1, Ying Li2, Di Wen3, Maodong Liu2, Yuteng Ma1 and Bin Cong3 Abstract Background: We sought to confirm that neutrophil gelatinase-associated lipocalin (NGAL) protects against apoptosis during endotoxemia. Methods: Endotoxemia was induced in rats with lipopolysaccharide (LPS; 3.5 mg/kg) and serum creatinine (SCr), urinary NGAL (uNGAL), renal histopathology confirmed acute kidney injury (AKI). Renal caspase 3 and NGAL were assayed with immunohistochemistry 6 h later. A HK-2 cell model was used in which NGAL and caspase 3 mRNA were evaluated by qRT-PCR within 6 h after LPS (50 μM) treatment, and correlations were studied. NGAL and caspase 3 mRNA expression were measured after delivering NGAL siRNA in HK-2 cells and apoptosis was measured with TUNEL and flow cytometry. Results: SCr and uNGAL were significantly increased after LPS treatment and renal morphology data indicated AKI and renal tubular epithelial cell apoptosis. Caspase 3 and NGAL were predominantly expressed in the tubular epithelial cells and there was a correlation between caspase 3 and NGAL protein (r = 0.663, p = 0.01). In vitro, there was a strong correlation between caspase 3 and NGAL mRNA in LPS-injured HK-2 cells within 24 h (r = 0.448, p < 0.05). Suppressing the NGAL gene in HK-2 cells increased caspase 3 mRNA 4.5-fold and apoptosis increased 1.5-fold after LPS treatment. Conclusions: NGAL is associated with caspase 3 in renal tubular cells with endotoxin-induced kidney injury, and may regulate its expression and inhibit apoptosis. Keywords: Neutrophil gelatinase associated lipocalin, Acute kidney injury, Apoptosis, siRNA, Lipopolysaccharide in body fluids by damaged kidney tissues and initial work suggests that NGAL may be a promising indi- cator of septic AKI [6]. © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Han et al. BMC Nephrology (2018) 19:168 https://doi.org/10.1186/s12882-018-0977-3 Han et al. BMC Nephrology (2018) 19:168 https://doi.org/10.1186/s12882-018-0977-3 Background AKI occurs for more than half of ICU patients, with sepsis being the most common trigger, and the lack of sensitive and specific biomarkers to confirm renal cell injury increases mortality for septic AKI [1–3]. Recently, using genomic technology, transcriptome and proteome analysis, researchers identified NGAL as an acute phase protein (APP) and an early biological marker for AKI [4, 5]. Many studies of AKI in critically ill pa- tients have suggested the diagnostic value of NGAL which is rapidly expressed, synthesized and secreted Whether this APP can reduce kidney injury or reverse damage is unknown because few studies have been pub- lished to describe renal NGAL expression during AKI. Previous work suggests that [4, 7] damaged rat kidney expresses NGAL significantly in early stages of ischemic AKI, and NGAL mRNA increased 1000 times. In our early AKI rat model, renal epithelial cells after lipopoly- saccharide (LPS) administration were edematous and apoptotic and NGAL mRNA expression was increased [8]. However, the function of NGAL is unclear, so we in- vestigated renal epithelial cell death to explore the Correspondence: yinglihebei2016@163.com 2Department of Nephropathy, The Third Hospital of Hebei Medical University, Shijiazhuang 050051, China Full list of author information is available at the end of the article * Correspondence: yinglihebei2016@163.com 2Department of Nephropathy, The Third Hospital of Hebei Medical University, Shijiazhuang 050051, China Full list of author information is available at the end of the article * Correspondence: yinglihebei2016@163.com 2Department of Nephropathy, The Third Hospital of Hebei Medical University, Shijiazhuang 050051, China Full list of author information is available at the end of the article Page 2 of 10 Page 2 of 10 Han et al. BMC Nephrology (2018) 19:168 relationship between NGAL and apoptosis in a rat model and an HK-2 cell model of LPS-induced injury. Beijing, China) and reading changes in absorbance over 40 s in experimental samples relative to standard. relationship between NGAL and apoptosis in a rat model and an HK-2 cell model of LPS-induced injury. uNGAL measurements Previously, urinary NGAL (uNGAL) may reflect kidney injury more than plasma NGAL (pNGAL) [8]. Thus, urine was studied with the aid of metabolic cages. uNGAL was measured using a commercially available ELISA kit (CSB-E09409r, Cusabio Biotech, Wuhan, China), according to the manufacturer’s instructions. Previously, urinary NGAL (uNGAL) may reflect kidney injury more than plasma NGAL (pNGAL) [8]. Thus, urine was studied with the aid of metabolic cages. uNGAL was measured using a commercially available ELISA kit (CSB-E09409r, Cusabio Biotech, Wuhan, China), according to the manufacturer’s instructions. Immunohistochemistry and histological scoring NGAL and caspase 3 were evaluated by IHS. Renal tis- sues were fixed in 10% formalin for 24 h and subse- quently embedded in paraffin. HIS was performed on 4-μM renal sections using anti-NGAL and anti-active caspase 3 (Sigma). Slides were developed using HRP-labeled secondary antibody (Dako Denmark, Shanghai, China) and DAB (Sigma). All HIS analyses were repeated at least three times and representative im- ages are presented. Quantification of IHS was assessed in the cortex and cortico-medullary area: positive cells were counted in a high-power field (HPF, 20× magnifica- tion) or ten to fifteen high-power fields (20×) and images were obtained for each slide. Positive areas were mea- sured using ImageJ software (NIH, Bethesda, MD) [10]. Results are shown as positive areas as a percent of the total area analyzed. Methods Histopathology was conducted in kidney samples to de- termine the time-course of renal micro-morphological injury in the LPS-induced endotoxemic rats. Each kidney sample (one quarter from both the control and experi- mental groups) was fixed in 4% paraformaldehyde, dehy- drated in graded ethanol and embedded in paraffin as previously described [9]. Each paraffin block was proc- essed into 5-μm-thick slices and was H&E stained. A portion (~ 1 mm3) of renal cortex from each rat was fixed in 2.5% glutaraldehyde diluted in 0.066 M phos- phate buffer (pH 7.4) for 24 h. Samples were then dehy- drated in a graded ethanol series and embedded in Epon 812 resin at 60 °C for 48 h. Thin sections (50 nm) were then double-stained with uranyl acetate and lead citrate and were observed and photographed with a TEM oper- ated at 80 kV. The epithelial layer was examined and photographed with a TEM (Hitachi, H-7500, TEM) at a magnification of 5000×. Experimental groups In vivo experiments were conducted on male Sprague-Dawley rats (200 ± 20 g, 7–8 weeks old). They were purchased from the Experimental Animal Center of Hebei Medical University (Shijiazhuang, China) and maintained in a specific pathogen-free environment in our facility. Rats were fed freely with standard food and water and were cared for in accordance with the Local Committee of Animal Use and Protection of Hebei Medical University, China. Animals were housed with 12-h light-dark cycles and were acclimated for at least 1 week before experimentation. Because the response to LPS depends on temperature, the ambient temperature was set at 24 °C. Endotoxemia and AKI were induced in rats with LPS (Escherichia coli 0111: B4, Sigma, St. Louis, MO). In our previous vivo study [8], we observed that LPS-induced the upregulation of renal NGAL mRNA from 3 to 12 h after treatment compared with controls (p < 0.001). At peak expression of NGAL mRNA in- creased 260-fold (6 h post LPS). Thus, we selected 20 rats to evaluate the relationship of NGAL and kidney in- jury, rats were randomized into 2 groups by medical laboratory technicians (n = 10 rats/group). Group 1 in- cluded controls (Con) treated with isometric sterile sa- line. Group 2 was an endotoxin induced AKI group (sAKI) treated with LPS (3.5 mg/kg, ip), which was used previously [8], due to its ability to induce moderate endotoxemia in rats. Because expression of NGAL mRNA peaked at 6 h in rat renal epithelia cells, we se- lected the 6 h time point post-LPS treatment to evaluate NGAL and kidney injury. Urine was gathered using metabolic cages (Beijing, China) and supernatant was obtained. After 6 h of LPS or saline administration, all rats were anaesthetized with sodium pentobarbital (60 mg/kg ip), and 2 mL of blood was obtained by car- diac puncture and processed to obtain serum which was frozen at −80 °C for later analysis of SCr. Kidneys were harvested for hematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM) and immuno- histochemical staining (IHS). When tissue had been obtained, the animals were euthanized by dislocation of the cervical spine under deep anesthesia. Investigators were blinded to group allocation during modeling and analysis. Evaluating renal function HK-2 human renal proximal tubular epithelial cells were supplied from Shanghai Bioleaf Biotech (Shanghai, China). They were maintained at 37 °C in 5% CO2 with DMEM/F12 (Gibco Grand Island, NY) containing 10% Renal function measured with serum creatinine was assessed in Con and sAKI groups using a colorimetric assay (Creatinine Assay Kit; Biosino Bio-Technology, Han et al. BMC Nephrology (2018) 19:168 Page 3 of 10 Page 3 of 10 fetal bovine serum (FBS) and penicillin (100 units/ mL)-streptomycin (0.1 mg/mL) mixture (PAA, Strasse, Pasching, Austria) on plastic dishes. Cells were cultured in a humidified atmosphere at 37 °C with 5% CO2, and passaged twice per week. To minimize age-dependent variation, cells from passages 18–22 were used. antibiotics in the medium for 24 h. Orifice plates were divided into four groups: Con, LPS, siRNA and siRNA + LPS and three wells per group were used. Con and LPS-treated cells were incubated with medium, and siRNA and siRNA + LPS-treated cells were grown and transfected with 6 pM siRNA using Stealth RNAi siRNA Duplex Oligoribonucleotides and RNAiMAX ((Invitro- gen, Carlsbad, CA) according to the manufacturer’s in- structions. Cells were incubated for 36 h and then placed in D-MEM/F12 medium with LPS (50 μM) which was replaced in LPS and siRNA + LPS groups for 3 h and serum-free medium was used in Con and siRNA groups. To quantify NGAL and caspase 3 mRNA expression, RNA was harvested and cDNA was synthesized. Measurement of apoptosis using TUNEL Measurement of apoptosis using TUNEL The TUNEL procedure was applied to kidney sections to detect DNA fragmentation as an index of apoptosis. Counterstaining was performed with DAPI dye (1 μg/ mL, Kirkegaard Perry Laboratories, Tokyo, Japan). Paraf- fin sections of 3 μm thick fixed with 4% paraformalde- hyde in PBS were stained with a TMR red in situ Cell Death Detection Kit (Roche, Basel, Switzerland). Depar- affinized HK-2 cells were seeded at 1 × 105/mL on a glass coverslip which was placed in advance in 6-well plates, transfected and treated with LPS (50 μM) as pre- viously described, and LPS was applied for 6 h. Medium was removed and cells were fixed with 4% paraformalde- hyde for 30 min at room temperature, and then cleaned with PBS twice. Next, cells were incubated with 3% H2O2 methanol solution at room temperature for 30 min, and cleaned with PBS twice. HK-2 cells were in- cubated in the permeabilization solution (0.1% TritonX-100 in 0.1% sodium citrate) for 2 min on ice. Cells were incubated with TUNEL reaction mixture for 60 min at 37 °C in the dark and sections were exposed to DAPI dye for 4 min in the dark. Finally, sections were mounted with VectaShield (Vector Laboratories, Orton Southgate, UK). TUNEL-positive cells were counted in 5 Quantitative real-time PCR (rt-qPCR) q HK-2 cells were seeded in 6-well plates at a density of 2 × 105 cells per well and were exposed to LPS of 50 μM for 24 h. Cells were harvested at 1, 3, 6, 12, and 24 h after LPS-treatment, and then total RNA was extracted with TriZol Reagent (Invitrogen, Carlsbad, CA) accord- ing to the manufacturer’s instructions. RNA was mea- sured using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE), and com- plementary DNA (cDNA) was synthesized from total RNA (500 ng) using a PrimeScriptRT regent Kit (Takara Biotechnology, Dalian, China) according to kit instruc- tions. Subsequently, cDNA was subjected to real-time PCR using Power SYBR Green PCR Master Mix (Takara Biotechnology, Dalian, China). Each real-time PCR reac- tion consisted of 2 μL of diluted RT product, 10 μL SYBR Green PCR Master Mix and 250-nm specific pri- mer pairs in a total volume of 20 μL. Reactions were per- formed on a 7500 real-time PCR System (Applied Biosystems, Foster, CA) for 40 cycles (95 °C for 5 s, 60 ° C for 35 s) after an initial 30 s incubation at 95 °C. PCR products were separated by 2% agarose gel electrophor- esis, illuminated with UV light and imaged to assess amplification. Fold changes in mRNA of each gene was calculated using the ΔΔCt method, with the housekeep- ing gene, GAPDH, as an internal control. NGAL, cas- pase 3 and GAPDH mRNA expression measured using primer sets as indicated in Table 1. Apoptotic assessment HK-2 cells were seeded, transfected and treated with LPS in 6-well plates as previously mentioned, but LPS treatment was 6 h to observe apoptosis which was mea- sured using an annexin V-FITC/ PI staining kit and flow cytometry (Becton Dickinson, San Jose, CA). After incu- bation, cells were washed twice with PBS and cell dens- ity was set at 1 × 106/mL with precooling Hank’s Balanced Salt Solution (HBSS). Then cells were incu- bated with fluorescein-conjugated annexin V and PI in the dark for 15 min at room temperature. Stained cells (1 × 105 cells/sample) were analyzed by flow cytometry and apoptosis was quantified. Results After 6 h of LPS injection, blood was collected to meas- ure SCr which increased almost 3.4-fold in the sAKI group compared to Con group (Fig. 1a). Renal morph- ology at the same time point indicated damage featuring severe tubular cell edema, cellular infiltrate and hyperemia in LPS-treated rats (Fig. 1b). Based on changes in SCr and renal histology, we confirmed that we established an LPS induced acute kidney injury ani- mal model. NGAL mRNA and caspase 3 mRNA were evaluated by qRT-PCR in HK-2 cells after LPS (50 μM) treat- ment. NGAL mRNA was significantly increased within 6 h compared with controls (LPS 1 and 3 h groups, p < 0.001; LPS 6 h group, p < 0.01), and Fig. 1 a SCr in rats subjected to LPS at 6 h. The data show SCr of Con and sAKI, with means ± SD values/group obtained by colorimetric assay (μM/L). SCr increased in rats with sAKI. SCr: serum creatinine, LPS: lipopolysaccharide. p < 0.01, relative to the Con group. b Renal histological injury observed under light microscopy at 6 h after LPS treatment (H&E, 200×). Histopathlogical changes included renal tubular epithelial swelling and inflammatory cell infiltration without glomerular injury. c uNGAL in rats subjected to LPS at 6 h. Data show respective uNGAL of Con and sAKI, with means ± SD values/group obtained by ELISA (ng/mL). uNGAL was significantly increased in rats with sAKI. uNGAL: urine NGAL, LPS: lipopolysaccharide. p < 0.001, relative to the con group Fig. 1 a SCr in rats subjected to LPS at 6 h. The data show SCr of Con and sAKI, with means ± SD values/group obtained by colorimetric assay (μM/L). SCr increased in rats with sAKI. SCr: serum creatinine, LPS: lipopolysaccharide. p < 0.01, relative to the Con group. b Renal histological injury observed under light microscopy at 6 h after LPS treatment (H&E, 200×). Histopathlogical changes included renal tubular epithelial swelling and inflammatory cell infiltration without glomerular injury. c uNGAL in rats subjected to LPS at 6 h. Data show respective uNGAL of Con and sAKI, with means ± SD values/group obtained by ELISA (ng/mL). uNGAL was significantly increased in rats with sAKI. uNGAL: urine NGAL, LPS: lipopolysaccharide. p < 0.001, relative to the con group Fig. 1 a SCr in rats subjected to LPS at 6 h. Statistical analysis All experiments were performed in duplicate and re- peated at least three times. Data are expressed as means ± SEM. Group comparisons were performed using ANOVA (SPSS, v. 16.0, Chicago, IL). All groups were analyzed simultaneously with an LSD t-test and p < 0.05 was considered statistically significant. IHS in the sAKI group revealed that caspase 3 protein was predominantly expressed in the renal tubular epi- thelium and NGAL staining increased as well. No abnor- mal findings were observed in controls (Fig. 2b and c). Consistently, semi-quantitative analysis results showed significant differences between both groups (p < 0.001, Fig. 2d). IHS suggested a correlation between caspase 3 and NGAL proteins, and NGAL may increase or de- crease epithelial apoptosis (r = 0.663, p = 0.01). Gene silencing by siRNA siRNA transfections HK-2 cells were seeded in 6-well plates at a density of 2 × 105 cells per well without Table 1 The sequences of primers used for RT-PCR and NGAL siRNA gene sequence product(bp) NGAL TTGGGACAGGGAAGACGA 240 TCACGCTGGGCAACATTA Caspase3 GTTCATCCAGTCGCTTTGTGC 110 AAATTCTGTTGCCACCTTTCG β-actin TCGCGGGAGACCACCGACAC 258 GGGGTGTTGGGTCAGGTCTCTG siRNA UUUAGUUCCGAAGUCAGCUCCUUGG CCAAGGAGCUGACUUCGGAACUAAA Table 1 The sequences of primers used for RT-PCR and NGAL Han et al. BMC Nephrology (2018) 19:168 Han et al. BMC Nephrology (2018) 19:168 Page 4 of 10 Page 4 of 10 Urine sample was analyzed with ELISA. Data indicate that 6 h after LPS-treatment, uNGAL of sAKI group sig- nificantly increased, indicating pathological renal lesions in the early stage of acute rat endotoxemia (Fig. 1c). randomly selected fields (400× magnification) and percents were calculated against total DAPI-stained cells. Two independent observers blinded to experimental conditions performed counts and calculated average TUNEL-positive cells. Data were collected from more than 3 independent experiments performed in triplicate. randomly selected fields (400× magnification) and percents were calculated against total DAPI-stained cells. Two independent observers blinded to experimental conditions performed counts and calculated average TUNEL-positive cells. Data were collected from more than 3 independent experiments performed in triplicate. To confirm that LPS induced renal tubular epithelial apoptosis, proximal tubular epithelial cells of renal corti- ces were observed under TEM 6 h after LPS injection. Damage was evident and nuclei were apoptotic. Microvilli were visibly disordered and deficient, and we observed intracellular edema, impaired mitochondrial outer membranes, nuclear membrane contraction, and chromatin at the edge of apoptotic cells (Fig. 2a). Results The data show SCr of Con and sAKI, with means ± SD values/group obtained by colorimetric assay (μM/L). SCr increased in rats with sAKI. SCr: serum creatinine, LPS: lipopolysaccharide. p < 0.01, relative to the Con group. b Renal histological injury observed under light microscopy at 6 h after LPS treatment (H&E, 200×). Histopathlogical changes included renal tubular epithelial swelling and inflammatory cell infiltration without glomerular injury. c uNGAL in rats subjected to LPS at 6 h. Data show respective uNGAL of Con and sAKI, with means ± SD values/group obtained by ELISA (ng/mL). uNGAL was significantly increased in rats with sAKI. uNGAL: urine NGAL, LPS: lipopolysaccharide. *p < 0.001, relative to the con group Page 5 of 10 Han et al. BMC Nephrology (2018) 19:168 a b c d Fig. 2 a Renal tubular epithelial cell injury and apoptosis observed under TEM at 6 h after LPS treatment (TEM, 5000×). Arrows indicate disarrayed microvilli, mitochondrial ballooning, and unevenly distributed nuclear chromatin in the outer nuclear layer gathered toward the center. TEM: transmission electron microscopy, LPS: lipopolysaccharide. b Localization of NGAL protein expression in rat kidneys under light microscopy 6 h after LPS treatment. Con: Control group, sAKI-1: renal cortex of sAKI group, sAKI-2: renal medulla of the sAKI group. (original magnification 400×). NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide. c Localization of caspase 3 protein expression in rat kidneys under light microscopy at 6 h after LPS treatment. Con: Control group, sAKI-1: renal cortex of sAKI group, sAKI-2: renal medulla of the sAKI group. Original magnification 400×. LPS: lipopolysaccharide. d Semi-quantification of immunohistochemical staining for NGAL and caspase 3 in kidneys of Con and sAKI rats. Data are expressed as means ± SD. p < 0.001, relative to controls a b c b d d epithelial cell injury and apoptosis observed under TEM at 6 h after LPS treatment (TEM, 5000×). Arrows indicate disarrayed al ballooning, and unevenly distributed nuclear chromatin in the outer nuclear layer gathered toward the center. TEM: microvilli, mitochondrial ballooning, and unevenly distributed nuclear chromatin in the outer nuclear layer gathered toward the center. TEM: transmission electron microscopy, LPS: lipopolysaccharide. b Localization of NGAL protein expression in rat kidneys under light microscopy 6 h after LPS treatment. Con: Control group, sAKI-1: renal cortex of sAKI group, sAKI-2: renal medulla of the sAKI group. (original magnification 400×). NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide. Results c Localization of caspase 3 protein expression in rat kidneys under light microscopy at 6 h after LPS treatment. Con: Control group, sAKI-1: renal cortex of sAKI group, sAKI-2: renal medulla of the sAKI group. Original magnification 400×. LPS: lipopolysaccharide. d Semi-quantification of immunohistochemical staining for NGAL and caspase 3 in kidneys of Con and sAKI rats. Data are expressed as means ± SD. p < 0.001, relative to controls (p < 0.01). NGAL mRNA expression of siRNA + LPS-treated cells was also significantly suppressed com- pared to controls (p < 0.01) and less than 20% when compared to LPS cells (p < 0.001), but slightly higher than in siRNA cells (p > 0.05). Caspase 3 mRNA also in- creased significantly in LPS-treated cells (p < 0.01) and was not different than controls in siRNA-treated cells (p > 0.05). Caspase 3 mRNA of siRNA + LPS-treated cells increased compared to controls (p < 0.001) and was twice that of LPS-treated cells (p < 0.01; Fig. 4). decreased to baseline after 12 h (p > 0.05). Peak ex- pression of NGAL occurred 3 h after LPS treatment (Fig. 3a). caspase 3 mRNA expression was upregu- lated within 3 h after LPS administration (LPS 1 h group, p < 0.001; LPS 3 h group, p < 0.05) and de- creased to baseline after 6 h. Expression after 6 and 12 h was not different than controls (LPS 6 and 12 h groups, p > 0.05). Peak expression of caspase 3 mRNA occurred in the LPS 1 h group and was almost twice greater than controls (Fig. 3b). caspase 3 and NGAL mRNA were correlated (r = 0.448, p < 0.05). Flow cytometry measurement of necrosis and apoptosis were assayed and increased only slightly after siRNA transfection and did not differ from controls (p > 0.05). NGAL siRNA did not injure HK-2 cells. But necrosis and apoptosis in HK-2 cells after LPS treatment were significantly greater than in controls (p < 0.001). In We measured NGAL mRNA and caspase 3 mRNA ex- pression after NGAL siRNA transfection after LPS stimulation of HK-2 cells. NGAL mRNA expression was significantly increased in LPS cells (p < 0.001), and suppressed in siRNA-treated cells compared to controls Han et al. BMC Nephrology (2018) 19:168 Page 6 of 10 Fig. 3 a Expression of NGAL mRNA in the LPS-treated HK-2. Results Data are means ± SD of three separate experiments in duplicate. There was a 2.2-fold increase in NGAL mRNA expression 1 h after LPS treatment, which increased to 3.2-fold at 3 h and then decreased to 1.8-fold at 6 h. NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide. p < 0.01, *p < 0.001, relative to the control group. b Expression of caspase 3 mRNA in the LPS-treated HK-2. Data are means ± SD of three separate experiments in duplicate. There was a 2.02-fold increase in NGAL mRNA expression at 1 h after LPS treatment, and this decreased to 1.3-fold at 3 h. p < 0.01, *p < 0.001, relative to controls. NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide Fig. 3 a Expression of NGAL mRNA in the LPS-treated HK-2. Data are means ± SD of three separate experiments in duplicate. There was a 2.2-fold increase in NGAL mRNA expression 1 h after LPS treatment, which increased to 3.2-fold at 3 h and then decreased to 1.8-fold at 6 h. NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide. p < 0.01, *p < 0.001, relative to the control group. b Expression of caspase 3 mRNA in the LPS-treated HK-2. Data are means ± SD of three separate experiments in duplicate. There was a 2.02-fold increase in NGAL mRNA expression at 1 h after LPS treatment, and this decreased to 1.3-fold at 3 h. p < 0.01, *p < 0.001, relative to controls. NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide siRNA + LPS-treated cells, necrosis was significantly greater compared to controls (p < 0.001), but there was no difference compared to LPS-treated cells (p > 0.05). Apoptosis in siRNA + LPS-treated cells was significantly greater than in the other three groups (Fig. 5a and b). Furthermore, the effect of NGAL siRNA on tubular cell apoptosis according to TUNEL staining showed similar results. There were few TUNEL-positive cells in sham HK-2 cells with/without transfection, but many TUNEL-positive cells appeared in LPS-treated cells. siRNA + LPS-treated cells were the most apoptotic (Fig. 6). Thus, inhibition of NGAL aggravated endotoxin-induced renal tubular cell damage. siRNA + LPS-treated cells, necrosis was significantly greater compared to controls (p < 0.001), but there was no difference compared to LPS-treated cells (p > 0.05). Apoptosis in siRNA + LPS-treated cells was significantly greater than in the other three groups (Fig. 5a and b). Results Furthermore, the effect of NGAL siRNA on tubular cell apoptosis according to TUNEL staining showed similar results. There were few TUNEL-positive cells in sham HK-2 cells with/without transfection, but many TUNEL-positive cells appeared in LPS-treated cells. siRNA + LPS-treated cells were the most apoptotic (Fig. 6). Thus, inhibition of NGAL aggravated endotoxin-induced renal tubular cell damage. Discussion NGAL and matrix metalloproteinase-9 (MMP-9) was observed during a study of gelatinase (92 kDa) in neu- trophil granulocytes in 1993 by Kjeklsen’s group [3, 11]. Then the complete genome sequence of NGAL cDNA was cloned and identified in 1994 and 1997, respectively [12, 13]. Recently, using gene expression microarray Mishra and colleagues reported that NGAL gene expres- sion in the kidney increased in early stage AKI in animal models. Down-stream proteomics analysis also showed that in ischemic and nephrotoxic AKI, NGAL was the APP that increased when induced [4]. Ischemic AKI caused by cardiac surgery and kidney transplantation, Fig. 4 Expression of NGAL mRNA and caspase 3 mRNA in HK-2 after NGAL siRNA transfection. Data are means ± SD of three separate experiments in duplicate. p < 0.01, *p < 0.001, relative to controls. ##p < 0.01, ###p < 0.001, relative to the LPS group. NGAL: neutrophil gelatinase-associated lipocalin Fig. 4 Expression of NGAL mRNA and caspase 3 mRNA in HK-2 after NGAL siRNA transfection. Data are means ± SD of three separate experiments in duplicate. p < 0.01, *p < 0.001, relative to controls. ##p < 0.01, ###p < 0.001, relative to the LPS group. NGAL: neutrophil gelatinase-associated lipocalin Page 7 of 10 Han et al. BMC Nephrology (2018) 19:168 Fig. 5 a Apoptosis and necrosis of HK-2 after NGAL siRNA3 transfection. Flow cytometry data for HK-2 after transfection. Necrotic cells appear in upper right quadrant, and apoptotic cells appear in lower right quadrant. A, Con group; B, LPS group; C, siRNA group; D, siRNA+ LPS group. NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide. b Apoptosis and necrosis of HK-2 after NGAL siRNA3 transfection. Flow cytometry data for four groups. Data are means ± SD of three separate experiments in duplicate. NGAL: neutrophil gelatinase-associated lipocalin, p < 0.001, relative to apoptosis controls. ###p < 0.001, relative to necrosis controls Fig. 5 a Apoptosis and necrosis of HK-2 after NGAL siRNA3 transfection. Flow cytometry data for HK-2 after transfection. Necrotic cells appear in upper right quadrant, and apoptotic cells appear in lower right quadrant. A, Con group; B, LPS group; C, siRNA group; D, siRNA+ LPS group. NGAL: neutrophil gelatinase-associated lipocalin, LPS: lipopolysaccharide. b Apoptosis and necrosis of HK-2 after NGAL siRNA3 transfection. Flow cytometry data for four groups. Data are means ± SD of three separate experiments in duplicate. NGAL: neutrophil gelatinase-associated lipocalin, p < 0.001, relative to apoptosis controls. Discussion c TUNEL-positive cells observed under light microscopy, and apoptotic cells are dark brown (200×). d TUNEL-positive cells counted and expressed as means ± SD. TUNEL: terminal-deoxynucleoitidyl transferase mediated nick end labeling. p < 0.001, relative to the control group. ##p < 0.01, relative to the LPS group Fig. 6 Apoptotic cells in HK-2 after transfection observed by TUNEL. a HK-2 observed under light microscopy (200×). b HK-2 observed under a fluorescent microscope, apoptotic cells are fluorescent green (200×). c TUNEL-positive cells observed under light microscopy, and apoptotic cells are dark brown (200×). d TUNEL-positive cells counted and expressed as means ± SD. TUNEL: terminal-deoxynucleoitidyl transferase mediated nick end labeling. **p < 0.001, relative to the control group. ##p < 0.01, relative to the LPS group significantly decreased. Renal tubular epithelial apop- tosis was reduced as was kidney injury, proving that apoptosis was an important injurious mechanism during septic AKI [29]. mitochondria and decreases ATP generation and this is chiefly affected by intracellular changes in chemical in- formation [25]. Another includes tumor necrosis factor (TNF) and TNF receptor (TNFR), which are mainly af- fected by extracellular stimulating information [26]. Cas- pases are proteases associated with apoptosis. Typically, they only exist in cells as cysteine proteases with low ac- tivity. When apoptosis was initiated by mitochondria or the TNF/TNFR pathway, caspase is activated by prote- olysis to form apoptotic caspases 2, 3, 6, 7, 8, 9, and 10. A final apoptotic protease, caspase 3 can degrade intra- cellular proteins, creating an apoptotic body [27]. g p In our study, LPS was given as described in the Methods and AKI was established as evidenced by in- creased SCr and uNGAL. Histologically, injury was also confirmed by inflammatory cell infiltration in the renal interstitium, parenchymal injury in renal tubular epithe- lial cells in the renal cortex, cloudy swelling of the cyto- plasm, the disappearance of the brush border, diminished lumen, and diminished or missing ligaments. Electron microscopy revealed apoptosis of renal tubular epithelial cells but not necrosis. Epithelial cells had contracted nuclear membranes and chromatin margin- ation of apoptosis. In our study, septic AKI in rats did not cause necrosis of renal tubular epithelial cells but apoptosis did occur. Thus, apoptosis is an important pathological injury mechanism for septic AKI, and these data agree with that of Guo’s group [29]. Discussion ###p < 0.001, relative to necrosis controls renal proximal tubular cells from hypoxic injury and death by up-regulating hemoxygenase [7, 15, 16]. Mishra’s group suggested that exogenous NGAL could protect renal proximal tubular cells, alleviate ischemia-reperfusion injury and inhibit apoptosis after injury [17]. Currently, NGAL during septic AKI is thought to have a protective effect but the mechanism is not understood; however, necrosis or apoptosis, which are consequences of irreversible injury [18–20] can be caused by numerous factors, and can co-occur. Whether necrosis or apoptosis predominates depends on the strength of the stimulating factor and the biological cel- lular characteristics [18, 21, 22]. Previous studies indi- cated that during AKI, apoptosis occurred with acute tubular necrosis (ATN). Lieberthal’s group has reported that cis-platinum to stimulate renal tubular epithelial cells induced apoptosis [23]. Furthermore, ATN has been verified in many ischemic AKI animal models, but this has not detected with a histologic examination of septic AKI, indicating that apoptosis may be the main cause of renal injury during septic AKI [10, 24]. nephrotoxic AKI caused by contrast agent, and septic AKI in critically ill patients all suggested that NGAL was a good biomarker for AKI early-stage diagnosis [6]. However, the function of NGAL expression in these events is unclear. Previous studies indicated that as a lipocalin, NGAL could combine with and stabilize hydrophobic small mo- lecular substances. Many human cancer cells can secrete NGAL. Yan’s group reported that NGAL could stabilize MMP-9 in neutrophilic granulocytes. NGAL covalently bind with MMP-9, and inhibited the degradation of MMP-9 and increased its activity. Also, MMP-9 pro- moted growth, infiltration and migration of carcinoma tissues by degrading the basement membrane and extra- cellular matrix, releasing vascular endothelial growth factors and promoting neonatal angiogenesis. Therefore, NGAL was considered to correlate with poor prognosis in cancer [14]. Morik and Schmidt reported that NGAL could generate an NGAL:ironophore, an iron complex which inhibited bacterial uptake of iron causing a bac- teriostatic effect, and promoted kidney mesenchymal cells during the embryonic period to differentiate into proximal tubular cells. Furthermore, it also protected Two mechanisms are said to cause apoptosis. First, a stressor (hypoxia or oxidative stress) stimulates Han et al. BMC Nephrology (2018) 19:168 Page 8 of 10 Fig. 6 Apoptotic cells in HK-2 after transfection observed by TUNEL. a HK-2 observed under light microscopy (200×). b HK-2 observed under a fluorescent microscope, apoptotic cells are fluorescent green (200×). AKI: Acute kidney injury; APP: Acute phase protein; ATN: Acute tubular necrosis; cDNA: Complementary DNA; FBS: Fetal bovine serum; H&E: Hematoxylin and eosin; HBSS: Hank’s Balanced Salt Solution; HPF: High- power field; IHS: Immunohistochemical staining; LPS: Lipopolysaccharide; MMP-9: Matrix metalloproteinase-9; NGAL: Neutrophil gelatinase-associated lipocalin; pNGAL: Plasma NGAL; RNAi: RNA interference; rt-qPCR: Real-time PCR; sAKI: LPS-induced AKI group; SCr: Serum creatinine; TEM: Transmission electron microscopy; TNF: Tumor necrosis factor; TNFR: TNF receptor; uNGAL: Urinary NGAL Competing interests The authors declare that they have no competing interests. Discussion Also, in injured renal tubular epithelial cells, NGAL and protein were significantly increased and correlated, indicating that early septic AKI involves NGAL up-regulation in renal tubular epithelial cells that is related to apoptosis. The meaning of this association, however, is unclear. Guo’s group has suggested that apoptosis occurs in septic AKI induced by LPS and blood urea nitrogen is increased. About 3 h after LPS treatment, apoptotic cells were present in kidney tissues, and apoptosis was main- tained for 48 h. In TNFR1 knockout mice, after the same treatment, apoptosis in renal tubular epithelial cells was significantly decreased, and kidney injury was alleviated. Therefore, in sepsis, LPS in kidney tissues mediated apoptosis by TNF/TNFR1, and apoptosis of epithelial cells was an important pathological pathway for kid- ney injury [28]. Also, LPS treatment increased blood urea nitrogen and renal caspase 3 activity increased. After a caspase 3 inhibitor was applied activity Han et al. BMC Nephrology (2018) 19:168 Han et al. BMC Nephrology (2018) 19:168 Han et al. BMC Nephrology (2018) 19:168 Page 9 of 10 Page 9 of 10 Using LPS to stimulate human proximal renal tubu- lar epithelial cells and HK-2 cells, to observe the rela- tionship between NGAL and caspase 3 at the genetic level, we found that 1–3 h after treatment, caspase 3 mRNA was significantly up-regulated and 1–6 h after LPS treatment, NGAL mRNA was up-regulated, indi- cating that apoptosis in HK-2 cells was initiated in a manner similar to rats with septic AKI and that NGAL and caspase 3 were linked. Authors’ contributions MH performed the experiments and data analysis, participated in the design of the study and drafted the manuscript. DW provided technical support. YL and BC conceptualized and designed the study and supervised the work. MDL and YTM provided intellectual input for the study and helped with the revision of the manuscript. All authors read and approved the final manuscript. All authors contributed to manuscript and approved the final version. Received: 13 March 2017 Accepted: 28 June 2018 Received: 13 March 2017 Accepted: 28 June 2018 Acknowledgements We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Availability of data and materials The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. p Specific siRNA with homologous sequences can si- lence target gene expression via RNA interference (RNAi) [30–32]. To study the biological effect of NGAL in renal epithelial cells, specific NGAL siRNA was applied to silence the NGAL gene and LPS stimulation was applied (siRNA + LPS group) to ob- serve changes in apoptosis. Caspase 3 mRNA was sig- nificantly increased compared to controls and the LPS-treated cells. Flow cytometry and TUNEL assay confirmed apoptotic cells in the siRNA + LPS was significantly greater than in other groups. Thus, when HK-2 cells were stimulated by LPS, intracellular NGAL was quickly synthesized and up-regulated by inhibiting caspase 3, which inhibited apoptosis to pro- tect HK-2 cells from death. With septic AKI kidney injury arose from factors including LPS, hypoxia, oxi- dative stress and inflammatory factors. If caspase 3 production can be inhibited, injured cells may survive and permit renal healing and protect renal function. Inhibition of caspase 3 by NGAL in injured epithelial cells may be a promising target for treating septic AKI. Funding This study was financially supported by the Applied Basic Research Key Project of Hebei Province(CN) (No.GL200809). The funding body played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Conclusions 1. 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https://openalex.org/W2051289805	https://europepmc.org/articles/pmc2782027?pdf=render	English	null	Inactivation of Mammalian Target of Rapamycin Increases STAT1 Nuclear Content and Transcriptional Activity in α4- and Protein Phosphatase 2A-dependent Fashion	Journal of biological chemistry/The Journal of biological chemistry	2,009	cc-by	11,940	Inactivation of Mammalian Target of Rapamycin Increases STAT1 Nuclear Content and Transcriptional Activity in 4- and Protein Phosphatase 2A-dependent Fashion□ S Jill A. Fielhaber1, Ying-Shan Han1, Jason Tan, Shuo Xing, Catherine M. Biggs, Kwang-Bo Joung, and Arnold S. Kristof2 From the Critical Care and Respiratory Divisions and Meakins-Christie Laboratories, Faculty of Medicine, McGill University, Montreal, Quebec H3A 1A1, Canada nd Respiratory Divisions and Meakins-Christie Laboratories, Faculty of Medicine, McGill University, 1A1 Canada Target of rapamycin (TOR) is a highly conserved serine/thre- onine kinase that controls cell growth, primarily via regulation of protein synthesis. In Saccharomyces cerevisiae, TOR can also suppress the transcription of stress response genes by a mecha- nism involving Tap42, a serine/threonine phosphatase subunit, and the transcription factor Msn2. A physical association between mammalian TOR (mTOR) and the transcription factor signal transducer and activator of transcription-1 (STAT1) was recently identifiedinhumancells,suggestingasimilarroleformTORinthe transcription of interferon--stimulated genes. In the current study, we identified a macromolecular protein complex composed ofmTOR,STAT1,theTap42homologue4,andtheproteinphos- phatase 2A catalytic subunit (PP2Ac). Inactivation of mTOR enhanced its association with STAT1 and increased STAT1 nuclear content in PP2Ac-dependent fashion. Depletion of 4, PP2A, or mTOR enhanced the induction of early (i.e. IRF-1) and late (i.e. caspase-1, hiNOS, and Fas) STAT1-dependent genes. The regulation of IRF-1 or caspase-1 by mTOR was independent of other known mTOR effectors p70 S6 kinase and Akt. These results describe a new role for mTOR and 4/PP2A in the control of STAT1 nuclear content, and the expression of interferon--sensi- tive genes involved in immunity and apoptosis. response to mitogens and changes in cellular metabolism. The effectsofmTORoncellgrowthinvolvethephosphorylationofp70 S6 kinase (S6K) and the translation inhibitor 4E-BP1, key regula- tors of ribosomal biogenesis and the initiation of protein synthesis (2). In contrast to its role in the initiation of translation, the current study focuses on mTOR as a regulator of mammalian gene transcription. g p Studies in Saccharomyces cerevisiae have revealed possible mechanisms by which mTOR might control mammalian tran- scription factors. TOR-regulated transcriptional control path- ways include ribosomal biogenesis, the nutrient deprivation response, and the stress response (3). TOR stimulates 35 S ribo- somal RNA expression and ribosomal biogenesis in nutrient- dependent fashion. Inhibition of TOR (e.g. rapamycin or amino acid depletion) reproduces a catabolic response in part by inhibiting rRNA synthesis. In the nutrient deprivation and stress responses, TOR controls the nuclear localization of key transcription factors by mechanisms that require its associated serine/threonine phosphatases (i.e. Pph21, Pph22, or Sit4) and their adaptor, Tap42 (3). THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 36, pp. 24341–24353, September 4, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 36, pp. 24341–24353, September 4, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. tory factor-1; hiNOS, human inducible nitric-oxide synthase; IFN-, inter- feron-; PKC, protein kinase C-; PP2Ac, protein phosphatase 2A catalytic subunit; S6K, p70 S6 kinase; siRNA, small interference RNA; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, gluta- thione S-transferase; PCA, protein fragment complementation assay; WT, wild type; CFP, cyan fluorescent protein; ECFP, enhanced CFP; YFP, yellow fluorescent protein. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Tables S1–S5. y 2 To whom correspondence should be addressed: McGill University Health Centre, Royal Victoria Hospital, 687 Pine Ave. W., L3.05, Montreal, Que- bec H3A 1A1, Canada. Tel.: 514-843-1664; Fax: 514-843-1686; E-mail: arnold.kristof@mcgill.ca. Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth Grant 5R01CA125436. This work was also facilitated by the McGill Univer- sity Health Centre Confocal Core Facility (S. Laporte) and funded by the Canadian Institutes for Health Research and by an American Thoracic Soci- ety/Lymphangioleiomyomatosis Foundation Award in partnership with the Tuberous Sclerosis Alliance and Tuberous Sclerosis Canada. □ tory factor-1; hiNOS, human inducible nitric-oxide synthase; IFN-, inter- feron-; PKC, protein kinase C-; PP2Ac, protein phosphatase 2A catalytic subunit; S6K, p70 S6 kinase; siRNA, small interference RNA; CHAPS, 3-[(3- cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GST, gluta- thione S-transferase; PCA, protein fragment complementation assay; WT, wild type; CFP, cyan fluorescent protein; ECFP, enhanced CFP; YFP, yellow fluorescent protein. * Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth Grant 5R01CA125436. This work was also facilitated by the McGill Univer- sity Health Centre Confocal Core Facility (S. Laporte) and funded by the Canadian Institutes for Health Research and by an American Thoracic Soci- ety/Lymphangioleiomyomatosis Foundation Award in partnership with the Tuberous Sclerosis Alliance and Tuberous Sclerosis Canada. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Tables S1–S5. 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed: McGill University Health Centre, Royal Victoria Hospital, 687 Pine Ave. W., L3.05, Montreal, Que- bec H3A 1A1, Canada. Tel.: 514-843-1664; Fax: 514-843-1686; E-mail: arnold.kristof@mcgill.ca. 3 The abbreviations used are: mTOR, mammalian target of rapamycin; STAT1, signal transducer and activator of transcription 1; IRF 1 interferon regula pp g 1 Both authors contributed equally to this work. g 3 The abbreviations used are: mTOR, mammalian target of rapamycin; STAT1, signal transducer and activator of transcription-1; IRF-1, interferon regula- * Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth Grant 5R01CA125436. This work was also facilitated by the McGill Univer- sity Health Centre Confocal Core Facility (S. Laporte) and funded by the Canadian Institutes for Health Research and by an American Thoracic Soci- ety/Lymphangioleiomyomatosis Foundation Award in partnership with the Tuberous Sclerosis Alliance and Tuberous Sclerosis Canada. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4 and Tables S1–S5. 1 Both authors contributed equally to this work. 2 To whom correspondence should be addressed: McGill University Health EXPERIMENTAL PROCEDURES Cell Culture and Cytokine Induction—A549, 2fTGH, and U3A cells were cultured as previously described (6, 12). HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin, 100 units/ml, and streptomycin, 100 g/ml. Cells were incu- bated without or with rapamycin (Biomol), 50 ng/ml, in serum- free or serum-containing media for 1 h before addition of IFN- (Roche Applied Science), 100 units/ml, for the indicated times. Transfection of siRNAs and Plasmids—A549 cells were trans- fected with 10–30 nM siRNA (siGENOME SMARTpool, Dhar- macon) directed against the indicated mRNAs (supplemental Table S1) using Dharmafect I, according to the manufacturer’s protocol. A non-targeting siRNA pool (siCONTROL) was used as a negative control. Specificity and potential off-target effects of the siRNA pool were tested using each of two individual siRNA duplexes per mRNA target (supplemental Table S1 and Fig. S3). For the expression of recombinant proteins, sub-con- fluent A549 or HEK 293T cells were incubated with serum-free medium and mammalian expression vectors, 0.5–1.0 g of plasmid DNA per 9.6-cm2 culture surface area, mixed with Lipofectamine 2000 or LTX (Invitrogen) in a 2:3 ratio for 5 h before replacement with fresh culture medium. Mammalian expression vectors pRK5 engineered to express Myc-tagged wild-type or dominant-negative (kinase-dead) mTOR (Dr. D. Sabatini, Massachusetts Institute of Technology), were ob- tained from Addgene. After 48–72 h, experimental protocols Cell Culture and Cytokine Induction—A549, 2fTGH, and U3A cells were cultured as previously described (6, 12). HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, penicillin, 100 units/ml, and streptomycin, 100 g/ml. Cells were incu- bated without or with rapamycin (Biomol), 50 ng/ml, in serum- free or serum-containing media for 1 h before addition of IFN- (Roche Applied Science), 100 units/ml, for the indicated times. GST Pulldown Assay—The cDNA encoding wild-type STAT1 (amino acids 1–750) was cloned by PCR into the Gate- way pDONR 221 Entry vector (Invitrogen) using human lung cDNA template and oligonucleotide primers listed in Table S3, and verified by automated sequencing. cDNAs were transferred to Gateway destination vector pDEST15 by recombination, before transformation of E. coli BL21 cells, and induction of GST-STAT1 protein synthesis with 2% arabinose as per the manufacturer’s protocol (Invitrogen). Purification of GST- STAT1 from crude bacterial lysates was performed by immo- bilization on glutathione-Sepharose (Amersham Biosciences), before recovery in elution buffer (50 mM Tris-HCl, pH 8, 40 mM glutathione). Regulation of STAT1 by mTOR were initiated, and lysates were prepared for detection of pro- tein or mRNA. mTORSTAT1 interactions were enhanced by Escherichia coli lipopolysaccharide and the cytokine interferon- (IFN-), a key inducer of genes involved in microbial killing, inflammation, and apoptosis. Although mTOR is a protein kinase, STAT1 did not appear to be its substrate. We subsequently proposed that, like Msn2 in yeast, mTOR might control the nuclear trafficking of STAT1 independent of its phosphorylation. Preparation of Cell Lysates for Detection of Proteins or Protein Complexes—Endogenous or recombinant proteins in whole cell or purified lysates were detected by Western blot analysis using antibodies listed in Table S2. Whole cell A549 or HEK 293T lysates were generated after washing once with cold phos- phate-buffered saline, and incubating for 15 min on ice in lysis buffer A (20 mM Tris, pH 8.0, 0.3% CHAPS, 1 mM EDTA, 10 mM -glycerophosphate, aprotinin, 10 g/ml, leupeptin, 10 g/ml, 1 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 100 M sodium orthovanadate). After freezing and thawing, cells were homogenized on ice and cleared (1,000 g for 5 min). Super- natants were further separated (16,000 g for 30 min) to gen- erate particulate-free lysates. For immunoprecipitation exper- iments, proteins (1 mg) from whole cell HEK 293T cells lysate were incubated with control IgG, anti-STAT1, or anti-Myc antibody, each 4 g, overnight at 4 °C before addition of 20 l of protein G-Sepharose for 1 h. Pellets were washed three times with phosphate-buffered saline containing 0.3% CHAPS before solubilization of bound proteins in SDS sample buffer for 5 min at 95 °C, separation by SDS-PAGE, and detection of bound pro- teins by Western blot. IFN- transcriptional programs are determined, in large part, by the control of STAT1 nuclear content, and binding of STAT1 to target gene promoters. Phosphorylation of its Tyr- 701 residue is required for IFN--induced STAT1 dimeriza- tion, accelerated translocation to the nucleus, and physical interaction with DNA-binding elements. STAT1 is also phos- phorylated at its Ser-727 residue by novel protein kinase C iso- forms (i.e. PKC and PKC) (7); this modification controls STAT1 transcriptional activity (8), as well as its nuclear export (9). The induction of early IFN--stimulated genes (e.g. IRF-1 and STAT1) is required for, and amplifies, the maximal expres- sion of late IFN--stimulated genes (10, 11). Regulation of STAT1 by mTOR Because mechanisms by which TOR controls gene transcrip- tion in yeast might be conserved, we hypothesized that inacti- vation of mTOR augments STAT1 nuclear localization by an 4- and PP2Ac-dependent mechanism. Here we demon- strate a dynamic association between mTOR, STAT1, PP2Ac, and 4 that is regulated by mTOR kinase activity. Inactivation of mTOR, by a mechanism that requires 4 and PP2Ac, increases the nuclear content of STAT1 and ampli- fies the induction of IFN--stimulated genes involved in inflammation and apoptosis. Preparation of Nuclear Lysates—A549 cell nuclear lysates were prepared as described previously (13). Briefly, cell pellets were resuspended in nuclear lysis buffer 1 (20 mM Tris, pH 7.5, 10 mM KCl, 1 mM dithiothreitol, 1 g/ml aprotinin, 1 g/ml leupeptin, 0.5 mM phenylmethylsulfonyl fluoride, 100 M sodium orthovanadate) before homogenization (Dounce, Pes- tle B) and centrifugation (1,100 g for 3 min). Supernatants were then centrifuged (16,000 g for 30 min) and used as cytosolic fractions, whereas pelleted nuclei were resuspended in 2 volumes of nuclear lysis buffer 2 (20 mM Tris, pH 7.5, 500 mM KCl, 1 mM dithiothreitol, 1 g/ml aprotinin, 1 g/ml leu- peptin, 0.5 mM phenylmethylsulfonyl fluoride, 100 M sodium orthovanadate) before freezing, thawing, and centrifugation for 30 min at 16,000 g. Inactivation of Mammalian Target of Rapamycin Increases STAT1 Nuclear Content and Transcriptional Activity in 4- and Protein Phosphatase 2A-dependent Fashion□ S Rapamycin blocks phosphorylation of the transcription factor Gln3, as well as its cytosolic scaffolding protein Ure2, permitting Gln3 translocation to the nucleus and the induction of nutrient discrimination pathway genes (4). During heat shock or osmotic stress, inactivation of TOR or Tap42 leads to sustained transcription of stress response genes by augmenting the nuclear content of the transcrip- tion factor Msn2 (5). TOR does not appear to affect the phos- phorylation of Msn2. Interactions between the mammalian homologues mTOR, 4, and the protein phosphatase 2A cat- alytic subunit (PP2Ac) have been described, but little is known regarding the mechanism by which TOR controls the induction of stress transcriptional responses in higher eukaryotes. The macrocyclic lactone rapamycin (Sirolimus, RapamuneTM), as well as its analogues temsirolimus (CCI-779, ToriselTM) and everolimus (RAD-001, CerticanTM), are approved for immuno- suppression after organ transplantation, treatment of renal cell carcinoma,andthepreventionofcoronaryarteryin-stentresteno- sis (1). Their only known target is mammalian target of rapamycin (mTOR),3 a highly conserved protein that controls cell growth in We recently reported a physical association between mTOR, the transcription factor signal transducer and activator of tran- scription-1 (STAT1), and the STAT1 kinase protein kinase C- (PKC) (6). In human lung epithelial carcinoma (A549) cells, g 3 The abbreviations used are: mTOR, mammalian target of rapamycin; STAT1, signal transducer and activator of transcription-1; IRF-1, interferon regula- g 3 The abbreviations used are: mTOR, mammalian target of rapamycin; STAT1, signal transducer and activator of transcription-1; IRF-1, interferon regula- JOURNAL OF BIOLOGICAL CHEMISTRY 24341 24341 SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 Regulation of STAT1 by mTOR The column was calibrated using Dex- tran blue (2000 kDa), aldolase (158 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa) molecular mass standards dissolved in Buffer A. Quantification of Protein Levels by Band Densitometry—Im- ages of Western blot films were acquired using an Alpha Imager (Alpha Inotech Corp.) and analyzed with Alpha Ease FC soft- ware (version 4.1.0). Band density was obtained using the spot density and autobackground functions. The means of band density values from multiple experiments were compared by Student’s t test. Plasmid vectors for protein fragment complementation assays (PCAs) were adapted for the Gateway cloning system and provided by Dr. S. Michnick (University of Montreal). These pcDNA3.1 destination vectors contain Gateway recom- bination sites inserted downstream from cDNAs for yellow flu- orescent protein fragments YF1 (amino acids 1–158) or YF2 (amino acids 159–239) (16) (supplemental Fig. S2). Wild-type PKC was cloned by PCR into the Gateway entry vector pDONR 221 using the primers and template listed in supple- mental Table S3. cDNAs encoding wild-type STAT1 or PKC were transferred to the pcDNA3.1-based PCA vectors by recombination. Plasmids, 1 g/9.6-cm2 dish surface area, were co-transfected in a 1:1 ratio (VF1-STAT1:VF2-PKC) by lipo- somal transfection (Lipofectamine LTX Reagent, Invitrogen) according to the manufacturer’s recommendation, before initi- ation of the indicated experimental protocols. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature, rinsed with phosphate-buffered saline, and mounted in Vectashield Mounting Medium containing the DNA-binding dye 4,6-diamidino-2-phenylindole (Vector Laboratories). Real-time PCR—RNA was extracted (Illustra RNAspin kit, Amersham Biosciences), and cDNA was generated by reverse transcription from 1 g of RNA (Superscript II, Invitrogen). TaqMan- or SYBR green-based real-time PCR was performed using 2 l of cDNA and Power SYBR green or TaqMan Univer- sal PCR master mix (ABI), as per the manufacturers’ instruc- tions. Primers are listed in supplemental Tables S4 and S5. PCR reactions were carried out for 45 cycles (ABI 7500 Real Time PCR System). Results are expressed as -fold induction in mRNA levels as calculated by the Ct method (14). y t Fluorescence Imaging of STAT1 or STAT1PKC Hetero- dimers—For detection of endogenous STAT1, A549 cells were fixed with 4% paraformaldehyde for 15 min at room tempera- ture before permeabilization with 0.2% Triton X-100 and incu- bation with mouse STAT1 antibody (Santa Cruz Biotechnol- ogy, Santa Cruz, CA), and then Alexa Fluor 568 conjugated to anti-mouse antibody (Invitrogen). Regulation of STAT1 by mTOR sample buffer, separated by SDS-PAGE, and detected by West- ern blot analysis. (Invitrogen) and the PCR product were ligated according to the manufacturer’s protocol (Invitrogen); the resulting con- struct was propagated in DB3.1 E. coli-competent cells (Invitrogen), and verified by automated sequencing. The cDNA encoding wild-type STAT1was transferred from pDONR 221-STAT1 WT to pcDNA3.1/nV5/ECFP-DEST by recombination. For experiments employing co-expression of PP2Ac isoforms, wild-type PP2Ac, or its constitutively active Y307F form, were cloned by PCR into Gateway entry vector pDONR 221 using plasmid templates PCDNA3.1- HA-PP2Ac WT and PCDNA3.1-HA-PP2Ac Y307F (Dr. P. Branton, McGill University), and primers listed in supplemen- tal Table S3. The dominant-negative PP2Ac form (L199P) inhibits endogenous PP2Ac function, and cannot bind or hydrolyze its substrates (15); this mutant was engineered by site-directed mutagenesis (Stratagene) using pDONR 221- PP2Ac WT as template and the following primers: forward, 5-ccaatgtgtgacttgccgtggtcagatccagatgac-3; reverse, 5-gtc- atctggatctgaccacggcaagtcacacattgg-3. The cDNAs encod- ing PP2Ac isoforms forms were transferred from their respective pDONR 221 plasmids to pcDNA3.1/nV5/DEST by recombination. A549 cells were transfected with pcDNA3.1/nV5/ECFP-STAT1 alone (1 g/9.6-cm2 dish sur- face area), or with empty vector or pcDNA3.1/nV5-PP2Ac isoforms. For co-expression experiments, STAT1 (0.7 g/9.6-cm2 dish surface area) and PP2Ac (0.3 g/9.6-cm2 dish surface area) mammalian expression plasmids were co- transfected. Purification of Endogenous Protein Complexes by Chro- matography—Whole cell lysates for ion-exchange and gel-fil- tration chromatography were prepared using lysis buffer A without detergent. HEK 293T whole cell homogenates were applied to a SP Sepharose column (Amersham Biosciences) before washing with 5 column volumes of buffer B (20 mM Tris- HCl, pH 8.0), and collection of unbound proteins (i.e. flow- through). Protein content was monitored by continuous UV absorption spectrometry. Bound proteins were eluted with a continuous NaCl gradient formed by automated mixing with buffer C (20 mM Tris-HCl, pH 8.0, 1 M NaCl), and collected in 1-ml fractions. Proteins in fractions from chromatographic peaks were detected by Western blot analysis or used for sub- sequent gel-filtration chromatography. For gel-filtration chromatography, 500-l aliquots of the partially purified mTORSTAT1PP2Ac-containing fractions generated by cation-exchange chromatography were applied to a Superose 6 column (Amersham Biosciences, 10-mm diame- ter/24-cm bed height), before perfusion with gel-filtration elu- tion buffer (Buffer A without detergent). Proteins or protein complexes were collected in 0.5-ml fractions and detected by Western blot analysis. EXPERIMENTAL PROCEDURES Purity was verified by Coomassie blue staining, and confirmed by Western blot analysis with antibodies against STAT1 or GST. Recombinant proteins, 10 g each, were added to 30 l of glutathione-Sepharose 4B (Amersham Biosciences), and mixed for 1 h at 4 °C, before addition of equal amounts of whole cell lysate from HEK 293T cells. Beads were washed with phosphate-buffered saline containing 1 mM phenylmethylsul- fonyl fluoride and 0.3% CHAPS. Proteins were eluted in SDS VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 24342 4342 JOURNAL OF BIOLOGICAL CHEMISTRY Regulation of STAT1 by mTOR For expression of enhanced cyan fluorescent protein (ECFP)-tagged STAT1, gateway desti- nation vector pcDNA3.1/nV5/ECFP-DEST was constructed by PCR-amplifying ECFP cDNA using pECFP-C1 (Clontech) as a template, and the following primers, each containing an EcoO109I restriction site: forward, 5-acaaagggcccaatggtga- gcaagggcga-3; reverse, 5-acaaagggcccttgtacagctcgtccat-3. EcoO109I digestion products of pcDNA3.1/nV5-DEST Fluorescence was detected by multitrack image acquisi- tion (ECFP: excitation 458 nm, emission 475 nm; YFP: exci- tation 512 nm, emission 529 nm; Alexa Fluor 568: excitation 578 nm, emission 603 nm; 4,6-diamidino-2-phenylindole: excitation 405 nm, emission 475 nm) using a Plan-Neofluar 40/1.3 oil differential interference contrast objective and Zeiss LSM 510 META scanning confocal microscope. Images were acquired at room temperature using the inte- grated Zeiss AxioCam HR digital sensor and LSM 510 soft- ware. Fluorescence intensity was assessed by measuring rel- SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 24343 JOURNAL OF BIOLOGICAL CHEMISTRY SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 Regulation of STAT1 by mTOR ative pixel density with ImageJ software (Version 1.38x, NIH), and images were displayed using a Zeiss LSM Image Browser (version 4.2.0.121, Carl Zeiss MicroImag- ing). Results are expressed as the ratio of nuclear to cytoplasmic pixel density. Regulation of STAT1 by mTOR FIGURE 1—continued the exception of PKC, monomeric forms of proteins in the mTORSTAT1 complex were absent in this cation exchange- purified fraction (i.e. Peak 2). In contrast to PP2Ac, mTOR, and PKC, 4 no longer co-eluted with STAT1 in cells exposed to IFN-. Although a small amount of S6K was recovered in Peak 2 (Fig. 1B), it did not co-fractionate with STAT1 after gel-filtra- tion chromatography (Fig. 1C). Therefore, the subset of mTOR that binds PKC and STAT1 co-elutes with PP2Ac and 4 and is distinct from that which regulates translation initiation or ribosomal biogenesis. The amount of STAT1 that co-elutes with mTOR and 4 represents a fraction of the input, because STAT1 is present in multiple complexes (Fig. 1, B and C). These data are consistent with variation in the amounts of mTOR- associated proteins that bound GST-STAT1 (Fig. 1A). To determine whether mTOR kinase activity regulates the association between STAT1 and mTOR, we exposed HEK 293T cells to serum-free media, rapamycin, or IFN-. Blockade of mTOR activity with rapamycin strengthened the association between STAT1 and mTOR as compared with baseline (Fig. 1D), but had no discernable effect on the association between STAT1 and PP2A or 4. As previously shown (6), IFN- increased the association between STAT1 and mTOR. Similar to findings in gel-filtration experiments (Fig. 1C), IFN- strengthened the association between STAT1 and PKC or PP2Ac. FIGURE 1—continued These results suggest that STAT1 is preferentially associated with inactivated mTOR. Consistent with the effect of rapamy- cin, the recombinant kinase-dead form of Myc-mTOR exhib- ited a stronger association with STAT1 than that with the wild type (Fig. 1E, lane 3 versus 1). IFN- enhanced the association between wild-type Myc-mTOR and STAT1 (Fig. 1E, lane 2 ver- sus 1) but failed to further increase that between kinase-dead Myc-mTOR and STAT1 (Fig. 1E, lane 4 versus 3). Despite an increased association with kinase-dead mTOR, the phospho- rylation of STAT1, as determined by phospho-specific antibod- ies or gel retardation, did not appear to be altered by rapamycin or expression of the kinase-dead mTOR mutant (supplemental Fig. S1). In vitro kinase assays using mTOR immunoprecipi- tates were not employed, because at least one other STAT1 kinase is present in the complex (i.e. PKC). Taken together, these data indicate that mTOR can be found in a dynamic mac- romolecular complex with STAT1, PKC, and the mTOR-as- sociated phosphatase subunits 4 and PP2Ac. RESULTS The Phosphatase Subunits 4 and PP2Ac Associate with mTOR and STAT1 in Mammalian Cells—We identified additional components of the mTORSTAT1 complex by affinity purification of proteins from HEK 293T whole cell lysates, a commonly used system for studying mTOR protein interactions. In GST pulldown assays, recombinant GST-STAT1, but not GST alone, associated with mTOR and PKC in HEK 293T lysates, as well as the mTOR-associated phosphatase subunit 4 (Fig. 1A). As expected, GST-STAT1 was strongly associ- ated with endogenous STAT1. Con- sistent with our previous work (6), IFN- led to a small but significant increase in the amount of mTOR or PKC that bound recombinant STAT1 (Fig. 1A, right panel). The association between 4 and STAT1 was reduced in lysates from cells exposed to IFN-. These results indicate that mTOR, PKC, and 4 can associate with recombinant STAT1; however, the levels of mTOR, PKC, or 4 bound to GST- STAT1 were a small fraction of the total cellular input (Fig. 1A, lanes 4 and 5 versus lanes 1 and 2), suggest- ing that the complex may not be abundant, or its assembly might require STAT1 post-translational modification. We therefore pro- ceeded to characterize the complex using endogenous proteins from intact cells. Endogenous proteins in HEK 293T whole cell lysates were sepa- rated by ion-exchange and gel-fil- tration chromatography. HEK 293T cells were serum-deprived for 1 h before incubation without or with IFN- for 30 min. After cation-ex- change chromatography (Fig. 1B), STAT1, and PKC were detected in VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 24344 JOURNAL OF BIOLOGICAL CHEMISTRY 24344 VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 FIGURE 1. STAT1 associates with mTOR, 4, and PP2Ac in a macromolecular complex. A, HEK 293T cells were exposed to serum-free medium for 1 h before incubation without or with IFN-, 100 units/ml for 30 min. Proteins in whole cell homogenates, 2.5 mg, were incubated with recombinant GST alone or GST-STAT1, each 10 g, bound to glutathione-Sepharose. Bound proteins and sample inputs (Inp) 0.5% of the cell lysate) were separated by SDS-PAGE and detected by Western blot analysis. Long (Inp-l) and short (Inp-s) exposures of the same gel are shown. Average band density for recovered mTOR S.E. from three independent experiments is shown (right panel, , p 0.05 versus control). B, proteins in whole cell homogenates, 40 mg, from cells serum-deprived cells (1 h) incubated without or with IFN- were separated by cation-exchange chromatography. Beginning at the first chromatographic peak (flow-through), 1-ml fractions were collected. A NaCl gradient was applied, beginning at 54 ml (0 mM NaCl). 30-l aliquots of fractions from the flow-through and the indicated three peaks were analyzed by Western blot analysis. C, samples, 500 l, from an mTORSTAT1-containing fraction (Peak 2, 70 ml) were resolved by gel-filtration chromatography, and the indicated proteins were detected by Western blot analysis. The gel-filtration column was calibrated with molecular mass markers (shown above). D, HEK 293T cells, or E, HEK 293T cells expressing Myc-tagged wild-type (WT) or kinase-dead (KD) mTOR, were serum-deprived for 1 h in the absence or presence of rapamycin before incubation without or with IFN- for 30 min. Proteins from whole cell lysates, 1 mg, were immunoprecipitated with STAT1 (D), -Myc (E), or normal IgG antibody before detection of protein G-Sepharose-purified proteins by Western blot analysis. STAT1 immunoprecipi- tates were run on separate gels for the detection of mTOR (left) and other mTORSTAT1 complex proteins (right), respectively. Average band density for recovered mTOR (mean S.E.) from three independent experiments is shown (right panel; , p 0.05 versus control). All gels are representative of 3–5 independent experiments. Regulation of STAT1 by mTOR Moreover, mTOR kinase activity regulates its association with STAT1. FIGURE 1—continued two chromatographic peaks at elution volumes of 60–64 ml (Peak 1, 110 mM NaCl) and 70–72 ml (Peak 2, 250 mM NaCl), respectively. mTOR, and its associated phosphatase subunit 4, was detected with PKC, STAT1, and PP2Ac, but only in frac- tions from Peak 2 (Fig. 1B). mLST8, a known mTOR-interact- ing adaptor protein, was also present in Peak 2. The peak elu- tion of mTOR-associated translational control proteins 4E-BP1 and S6K occurred at higher concentration of NaCl. Therefore, the transcription factor STAT1 co-purifies with mTOR, mLST8, PKC, PP2Ac, and 4, but not with the translational control factors 4E-BP1 or S6K. Fractions containing mTOR, STAT1, PP2Ac, and 4 (Peak 2) were next subjected to gel-filtration chromatography (Fig. 1C). In unstimulated cells, mTOR and mLST8 were detected at a molecular mass similar to that of thyroglobulin (669 kDa). In cells exposed to IFN-, STAT1, PKC, and PP2Ac were detected in high molecular weight fractions with mTOR; with FIGURE 1. STAT1 associates with mTOR, 4, and PP2Ac in a macromolecular complex. A, HEK 293T cells were exposed to serum-free medium for 1 h before incubation without or with IFN-, 100 units/ml for 30 min. Proteins in whole cell homogenates, 2.5 mg, were incubated with recombinant GST alone or GST-STAT1, each 10 g, bound to glutathione-Sepharose. Bound proteins and sample inputs (Inp) 0.5% of the cell lysate) were separated by SDS-PAGE and detected by Western blot analysis. Long (Inp-l) and short (Inp-s) exposures of the same gel are shown. Average band density for recovered mTOR S.E. from three independent experiments is shown (right panel, , p 0.05 versus control). B, proteins in whole cell homogenates, 40 mg, from cells serum-deprived cells (1 h) incubated without or with IFN- were separated by cation-exchange chromatography. Beginning at the first chromatographic peak (flow-through), 1-ml fractions were collected. A NaCl gradient was applied, beginning at 54 ml (0 mM NaCl). 30-l aliquots of fractions from the flow-through and the indicated three peaks were analyzed by Western blot analysis. C, samples, 500 l, from an mTORSTAT1-containing fraction (Peak 2, 70 ml) were resolved by gel-filtration chromatography, and the indicated proteins were detected by Western blot analysis. The gel-filtration column was calibrated with molecular mass markers (shown above). Regulation of STAT1 by mTOR Rapamycin Increases the Nuclear Content of mTORSTAT1 Component Proteins—STAT1 did not appear to be a kinase substrate for mTOR (6), and we reasoned that, like the yeast STAT1 content ob small magnitude, an marker protein Erp5 FIGURE2.TheeffectofrapamycinonnuclearSTAT1content.A549cellswereserumdeprivedfor1hwit or with rapamycin (50 ng/ml) before incubation without or with IFN-, 100 units/ml, for 0.5, 3, or 18 h. A, teinsweredetectedbyWesternblotanalysisaftergenerationofnuclearandcytosolicfractions.Gelsshow representative of six experiments. Average integrated band density 1000 (S.E. 5% of the mean for condition, †, p 0.05 rapamycin with IFN- versus IFN- alone; , p 0.05 versus control) is indicated b Western blots for nuclear phospho-STAT1, mTOR, PP2Ac, or 4. B, average integrated band density for nu STAT1 S.E. (, p 0.05 versus untreated control; †, p 0.05 rapamycin with IFN- versus IFN- alone). carcinoma (A549) cells, because HEK 293T cells do not express many IFN-- and STAT1-depend- ent genes (data not shown) that reg- ulate apoptosis or inflammation (6, 19, 20). A549 cells were serum-de- prived for 1 h in the absence or pres- ence of rapamycin, before incuba- tion with serum-free medium or IFN- for 0.5, 3, or 18 h, and prepa- ration of nuclear and cytosolic lysates. In cells exposed to IFN-, rapamycin further enhanced nuclear STAT1 levels (Fig. 2A, lanes 4 versus 2 and lanes 12 versus 10; Fig. 2B). The enhancing effect of rapamycin was observed for the Tyr-701- and Ser-727-phospho- rylated forms of STAT, indicating the accumulation of STAT1 in its transcriptionally active form. Sep- aration of nuclear and cytoplasmic proteins was confirmed by co-lo- calization of acetylated histone H3 and enolase, respectively. FIGURE2.TheeffectofrapamycinonnuclearSTAT1content.A549cellswereserumdeprivedfor1hwitho or with rapamycin (50 ng/ml) before incubation without or with IFN-, 100 units/ml, for 0.5, 3, or 18 h. A, p teinsweredetectedbyWesternblotanalysisaftergenerationofnuclearandcytosolicfractions.Gelsshown representative of six experiments. Average integrated band density 1000 (S.E. 5% of the mean for ea condition, †, p 0.05 rapamycin with IFN- versus IFN- alone; , p 0.05 versus control) is indicated bel Western blots for nuclear phospho-STAT1, mTOR, PP2Ac, or 4. B, average integrated band density for nucl STAT1 S.E. (, p 0.05 versus untreated control; †, p 0.05 rapamycin with IFN- versus IFN- alone). p y Incubation of cells with rapamy- cin alone for 0.5, 3, or 18 h was sufficient to increase the nuclear content of STAT1 in its unphos- phorylated form (Fig. Regulation of STAT1 by mTOR D, HEK 293T cells, or E, HEK 293T cells expressing Myc-tagged wild-type (WT) or kinase-dead (KD) mTOR, were serum-deprived for 1 h in the absence or presence of rapamycin before incubation without or with IFN- for 30 min. Proteins from whole cell lysates, 1 mg, were immunoprecipitated with STAT1 (D), -Myc (E), or normal IgG antibody before detection of protein G-Sepharose-purified proteins by Western blot analysis. STAT1 immunoprecipi- tates were run on separate gels for the detection of mTOR (left) and other mTORSTAT1 complex proteins (right), respectively. Average band density for recovered mTOR (mean S.E.) from three independent experiments is shown (right panel; , p 0.05 versus control). All gels are representative of 3–5 independent experiments. JOURNAL OF BIOLOGICAL CHEMISTRY 24345 24345 SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 Regulation of STAT1 by mTOR Regulation of STAT1 by mTOR Because rapamycin also en- hanced the association between STAT1 and PKC (Fig. 1D), we used PCA to test whether mTOR can regulate the nuclear levels of STAT1 complexes. Wild-type recombinant STAT1 and PKC, each linked to a complementary fragment of YFP, were expressed in A549 cells (Fig. 4B and supple- mental Fig. S2). After 48 h, physi- cal association between STAT1 and PKC resulted in the detec- tion of YFP fluorescence in the cytoplasm. Consistent with meas- urements of endogenous or CFP- tagged STAT1 (Fig. 3), incubation of cells with rapamycin alone for 1 h led to a 2.5-fold increase in STAT1PKC nuclear content. S d i ti l did t FIGURE 3. Rapamycin induces STAT1 translocation to the nucleus. A, untransfected A549 cells, or B, those expressing recombinant CFP-STAT1 alone, were incubated without or with serum in the absence or presence of rapamycin, 50 ng/ml, for 0 (Ctrl) or 1 h. Endogenous STAT1 (red) and CFP-STAT1 (cyan blue) were detected by indirect immunofluorescence, and direct fluorescence, confocal microscopy, respectively. Slides were mounted with solution containing the nuclear marker 4,6-diamidino-2-phenylindole (DAPI, navy blue). Sum- marized data (mean nuclear to cytoplasmic pixel density ratio S.E., n 3–5 cells per experiment) are shown below the images, and are representative of three independent experiments. , p 0.05 versus control. 1 h led to a 2.5-fold increase in STAT1PKC nuclear content. Serum deprivation alone did not alter the nuclear content of the STAT1PKC heterodimer. STAT1PKC translocation to the nucleus elicited by rapa- mycin was similar to that induced by IFN-, and slightly attenuated by the presence of serum. YFP fluorescence was not detected in cells co-transfected with empty vector con- trols (data not shown). lls per experiment) are shown p 0.05 versus control. plasmic ratio (Fig. 3A). Incubation of cells with rapamycin for 1 h also augmented endogenous (Fig. 3A) or CFP-STAT1 (Fig. 3B) levels in the nucleus. Serum deprivation minimally increased nuclear levels of recombinant CFP-STAT1, but not those of endogenous STAT1. In cells exposed to IFN-, rapa- mycin-induced changes in nuclear to cytoplasmic STAT1 ratio were difficult to quantify by fluorescence microscopy due to saturation of the nuclear signal (data not shown). Together, results from biochemical and imaging experiments indicate that the increased association between inactivated mTOR and STAT1 (Fig. 1, D and E) is accompanied by enhanced STAT1 nuclear content. Regulation of STAT1 by mTOR 2, A (lanes 3 and 11) and B). The nuclear accu- mulation of STAT1 was accompa- nied by elevated nuclear levels of mTOR, 4, and PP2Ac seen at all time points (Fig. 2A, lanes 3 and 11). We did not consistently ob- serve an inverse correlation between cytoplasmic and nuclear levels of mTORSTAT1-associ- ated proteins. These results sug- gest that a fraction of total cellular STAT1, mTOR, 4, and PP2Ac undergoes nuclear trafficking, and that inactivation of mTOR can increase the nuclear content of mTORSTAT1-associated proteins. FIGURE2.TheeffectofrapamycinonnuclearSTAT1content.A549cellswereserumdeprivedfor1hwithout or with rapamycin (50 ng/ml) before incubation without or with IFN-, 100 units/ml, for 0.5, 3, or 18 h. A, pro- teinsweredetectedbyWesternblotanalysisaftergenerationofnuclearandcytosolicfractions.Gelsshownare representative of six experiments. Average integrated band density 1000 (S.E. 5% of the mean for each condition, †, p 0.05 rapamycin with IFN- versus IFN- alone; , p 0.05 versus control) is indicated below Western blots for nuclear phospho-STAT1, mTOR, PP2Ac, or 4. B, average integrated band density for nuclear STAT1 S.E. (, p 0.05 versus untreated control; †, p 0.05 rapamycin with IFN- versus IFN- alone). enhanced the nuclear content of STAT1 (Fig. 2B), changes in nuclear Rapamycin Increases the Nuclear Content of mTORSTAT1 Rapamycin Increases the Nuclear Content of mTORSTAT1 Component Proteins—STAT1 did not appear to be a kinase substrate for mTOR (6), and we reasoned that, like the yeast TOR/Msn2 pathway (3), mTOR might control the nuclear con- tent of STAT1. To test for mTOR effects, we used transient exposure to its inhibitor rapamycin, because prolonged genetic or molecular manipulation of mTOR, or its endogenous sup- pressor TSC2, can alter STAT1 levels and activity (6, 18). Sub- sequent experiments were performed in lung epithelial adeno- STAT1 content observed by Western blot analysis were of small magnitude, and low levels of the endoplasmic reticulum marker protein Erp57 were detected in nuclear fractions (data not shown). To better determine the effect of mTOR inactiva- tion on constitutive STAT1 nuclear trafficking, we performed fluorescence confocal imaging of untransfected A549 cells, or those expressing wild-type cyan fluorescent protein (CFP)- tagged STAT1 (Fig. 3). As expected, IFN- led to a significant increase in nuclear STAT1 as measured by its nuclear to cyto- 24346 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 24346 VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 Regulation of STAT1 by mTOR FIGURE 3. Rapamycin induces STAT1 translocation to the nucleus. A, untransfected A549 cells, or B, those expressing recombinant CFP-STAT1 alone, were incubated without or with serum in the absence or presence of rapamycin, 50 ng/ml, for 0 (Ctrl) or 1 h. Endogenous STAT1 (red) and CFP-STAT1 (cyan blue) were detected by indirect immunofluorescence, and direct fluorescence, confocal microscopy, respectively. Slides were mounted with solution containing the nuclear marker 4,6-diamidino-2-phenylindole (DAPI, navy blue). Sum- marized data (mean nuclear to cytoplasmic pixel density ratio S.E., n 3–5 cells per experiment) are shown below the images, and are representative of three independent experiments. , p 0.05 versus control. STAT1 nuclear content requires PP2Ac activity, we co-expressed its constitutively-active (Y307F) or dominant-negative (L199A) form with CFP-STAT1 (Fig. 4A). Expression of the constitutively activeformblockedtherapamycin- dependent increase in nuclear CFP-STAT1 levels. Expression of dominant-negative PP2Ac caused a 2-fold increase in basal CFP- STAT1 levels. In contrast to untransfected (Fig. 3A) or empty vector-transfected cells (Figs. 3B, 4A, and 4B), rapamycin failed to further augment nuclear STAT1 levels in cells expressing domi- nant-negative PP2Ac. These results demonstrate that PP2Ac activity mediates the effects of mTOR inactivation on STAT1 nuclear trafficking. nuclear trafficking. Because rapamycin also en- hanced the association between STAT1 and PKC (Fig. 1D), we used PCA to test whether mTOR can regulate the nuclear levels of STAT1 complexes. Wild-type recombinant STAT1 and PKC, each linked to a complementary fragment of YFP, were expressed in A549 cells (Fig. 4B and supple- mental Fig. S2). After 48 h, physi- cal association between STAT1 and PKC resulted in the detec- tion of YFP fluorescence in the cytoplasm. Consistent with meas- urements of endogenous or CFP- tagged STAT1 (Fig. 3), incubation of cells with rapamycin alone for 1 h led to a 2.5-fold increase in STAT1PKC nuclear content. Serum deprivation alone did not alter the nuclear content of the STAT1PKC heterodimer. STAT1PKC translocation to the nucleus elicited by rapa- mycin was similar to that induced by IFN-, and slightly attenuated by the presence of serum. YFP fluorescence was not detected in cells co-transfected with empty vector con- trols (data not shown). nsfected A549 cells, or B, those um in the absence or presence T1 (cyan blue) were detected by py, respectively. Slides were lindole (DAPI, navy blue). Sum- ells per experiment) are shown , p 0.05 versus control. FIGURE4.mTORandPP2AcactivityregulateSTAT1nuclearcontent.A,A549cellsco-expressingCFP-STAT1 and PP2Ac isoforms (Y307F, constitutively active; L199P, dominant-negative) were incubated without or with serumintheabsenceorpresenceofrapamycin,50ng/ml,for0(Ctrl)or1h,beforedetectionofCFP-STAT1(cyan blue) by confocal fluorescence microscopy. B, STAT1 and PKC, each linked to complementary fragments of YFP were expressed in A549 cells. Physical interaction of the recombinant proteins leads to reconstitution of active YFP permitting detection by fluorescence microscopy. A549 cells expressing STAT1 and PKC PCA constructs were incubated without or with serum in the absence or presence of rapamycin, 50 ng/ml, or IFN-, 100 units/ml, for 1 h. Summarized data (mean nuclear to cytoplasmic pixel density ratio S.E., n 3–5 cells per experiment) are shown below representative images of CFP-STAT1 (cyan blue) or STAT1PKC heterodimers (yellow) and nuclei (navy blue), and represent four independent experiments. , p 0.05 versus control. We next determined whether the enhancing effect of mTOR blockade on IRF-1 or STAT1 expression requires 4 or PP2Ac (Fig. 5, D–F). Like rapamycin (Fig. 5, D and E, lanes 4 versus 2), siRNA-mediated depletion of 4 (Fig. 5D, lanes 6 versus 2) or PP2Ac (Fig. 5E, lanes 6 versus 2) increased IRF-1 protein levels in cells exposed to IFN-. Rapamycin failed to further increase IRF-1 protein induction in 4- or PP2Ac- deficient cells (lanes 8 versus 6). Phosphorylation of S6K at the PP2Ac/mTOR-sensitive T389 residue (23) was increased upon depletion of PP2Ac, indicating a reduction in PP2Ac activity (Fig. 5E, lanes 5 versus 1). Depletion of 4 also increased phos- phorylation of S6K, indicating that, as previously shown (24, 25), 4 can be a positive regulator of PP2Ac activity (Fig. 5D, lane 5 versus 1). IRF-1 induction was enhanced whether phos- phorylation of S6K was reduced (mTOR depletion (Fig. 5C)) or augmented (PP2Ac depletion (Fig. 5E)), suggesting that phosphorylation of S6K is not involved in mTOR regulation of STAT1. Depletion of mTOR, 4, or PP2Ac using each of two single siRNA duplexes reproduced the effects of pooled siRNAs on IRF-1 induction (supplemental Fig. S4). Consist- ent with elevated IRF-1 levels, depletion of 4 or PP2Ac enhanced STAT1 mRNA induction by 2- and 3-fold, respec- tively (Fig. 5F). Rapamycin failed to potentiate STAT1 mRNA induction in 4- or PP2Ac-deficient cells. Regulation of STAT1 by mTOR Taken together, complementary biochemical and imaging studies demonstrate that STAT1 nuclear levels are regulated by mTOR in PP2A-dependent fashion. Rapamycin can increase the nuclear content of unphosphorylated STAT1 in the absence of IFN-, or enhance the nuclear content of activated STAT1 in cells exposed to IFN-. We next deter- mined whether inactivation of mTOR, or the depletion of 4/PP2Ac phosphatase subunits, leads to enhanced induc- tion of STAT1-dependent genes. PP2Ac Mediates the Effect of Rapamycin on STAT1 Nuclear Content—In mammalian cells, mTOR and 4 associated with PP2Ac (21). To determine whether rapamycin effects on SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 2434 24347 SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 Regulation of STAT1 by mTOR FIGURE4.mTORandPP2AcactivityregulateSTAT1nuclearcontent.A,A549cellsco-expressingCFP-STAT1 and PP2Ac isoforms (Y307F, constitutively active; L199P, dominant-negative) were incubated without or with serumintheabsenceorpresenceofrapamycin,50ng/ml,for0(Ctrl)or1h,beforedetectionofCFP-STAT1(cyan blue) by confocal fluorescence microscopy. B, STAT1 and PKC, each linked to complementary fragments of YFP were expressed in A549 cells. Physical interaction of the recombinant proteins leads to reconstitution of active YFP permitting detection by fluorescence microscopy. A549 cells expressing STAT1 and PKC PCA constructs were incubated without or with serum in the absence or presence of rapamycin, 50 ng/ml, or IFN-, 100 units/ml, for 1 h. Summarized data (mean nuclear to cytoplasmic pixel density ratio S.E., n 3–5 cells per experiment) are shown below representative images of CFP-STAT1 (cyan blue) or STAT1PKC heterodimers (yellow) and nuclei (navy blue), and represent four independent experiments. , p 0.05 versus control. was pronounced 2 h after exposure to IFN- (Fig. 5B). Incubation of cells with rapamycin alone (i.e. in the absence of IFN-) did not appre- ciably increase IRF-1 mRNA or pro- tein levels (Fig. 5, A and B). To further demonstrate an inhib- itory role for mTOR, 4, or PP2Ac during the induction of IFN--stim- ulated genes, we transfected cells with pooled siRNA duplexes to maximize target mRNA and protein depletion while limiting potential off-target effects. In agreement, pooled siRNA targeting 4 or PP2Ac reduced mRNA levels more effectively than each of two individ- ual siRNA duplexes (supplemental Fig. S3, A and B). Moreover, neither pooled nor single siRNA duplexes significantly depleted the levels of other mRNAs encoding proteins in the mTOR pathways (supplemental Fig. S3, C and D). siRNA-mediated depletion of mTOR reproduced the effect of rapamycin on IRF-1 levels (Fig. 5C, lanes 4 versus 2). Regulation of STAT1 by mTOR Phospho- rylation of S6K at Thr-389 in mTOR-depleted cells was reduced (lanes 3 and 4), confirming inhibi- tion of mTOR activity. Regulation of STAT1 by mTOR In contrast, and as observed in previous studies (28), serum deprivation without or with rapamycin blocked the synthe- sis of 45 S rRNA; this response was not significantly altered by IFN- (Fig. 6B). The inhibitory effect of rapamycin on 45 S rRNA levels was preserved in STAT1-deficient human U3A cells (Fig. 6C), whereas Fas, hiNOS, or caspase-1 mRNA was undetectable (data not shown). As was the case for STAT1 and IRF-1, depletion of 4 or PP2Ac sig- nificantly increased hiNOS and caspase-1 mRNA levels in cells exposed to IFN- (Fig. 6D). Deple- tion of 4 or PP2Ac blocked the pamycin, indicating that both are mTOR on caspase-1 or hiNOS mRNA Unlike STAT1-dependent genes, inhibition of mTOR did not aug- ment levels of precursor ribosomal RNA (i.e. 45 S rRNA). In contrast, and as observed in previous studies (28), serum deprivation without or with rapamycin blocked the synthe- sis of 45 S rRNA; this response was not significantly altered by IFN- (Fig. 6B). The inhibitory effect of rapamycin on 45 S rRNA levels was preserved in STAT1-deficient human U3A cells (Fig. 6C), whereas Fas, hiNOS, or caspase-1 mRNA was undetectable (data not shown). As was the case for STAT1 and IRF-1, depletion of 4 or PP2Ac sig- nificantly increased hiNOS and caspase-1 mRNA levels in cells exposed to IFN- (Fig. 6D). Deple- tion of 4 or PP2Ac blocked the cing effect of rapamycin, indicating that both are ed for the effect of mTOR on caspase-1 or hiNOS mRNA IRF-1 expression in or presence of rapa- detection of IRF-1 or were transfected with h in the absence or ndicated proteins by th IFN- versus IFN-) N- in the absence or R. Changes in mRNA d, means of triplicate one in control-trans- FIGURE 5. Inhibition of mTOR, or depletion of 4/PP2Ac, augments STAT1 and IRF-1 expression in response to IFN-. A549 cells were incubated with serum-free medium in the absence or presence of rapa- mycin, 50 ng/ml, for 1 h before addition of IFN-, 100 units/ml, for 0.5, 6, or 18 h before detection of IRF-1 or STAT1 mRNA (A) or 0.5, 1, or 2 h before detection of IRF-1 protein (B). These data demonstrate a requirement for 4 and PP2Ac in the enhanc- eins leads to reconstitution of ressing STAT1 and PKC PCA rapamycin, 50 ng/ml, or IFN-, ty ratio S.E., n 3–5 cells per ) or STAT1PKC heterodimers p 0.05 versus control. Inactivation of mTOR Enhances the Induction of Early IFN- -stimulated Genes—Interferon regulatory factor-1 (IRF-1) is an early STAT1-dependent IFN response gene, the product of which, in a feed-forward positive feedback loop, directly acti- vates the STAT1 gene promoter (10). Newly synthesized STAT1 and IRF-1 then contribute to the induction of other late IFN--stimulated genes (22). Because rapamycin enhanced nuclear STAT1 protein levels (Figs. 2–4), we reasoned that inactivation of mTOR would augment IRF-1 expression in cells exposed to IFN-. A549 cells were serum-deprived in the absence or presence of rapamycin before incubation with IFN- for up to 18 h. Cells exposed to rapamycin and IFN- exhibited greater induction of IRF-1 mRNA (Fig. 5A, left panel; 13- versus 3-fold) and protein (Fig. 5B, lane 8 versus 6) than those exposed to IFN- alone (Fig. 5A; 13- versus 3-fold). Sub- sequent induction of STAT1 mRNA at 6 h, which is primarily regulated by IRF-1, was also significantly enhanced by rapamy- cin (Fig. 5A, right panel). Interestingly, the effect of rapamycin was less pronounced at 6 h, and then resumed at 18 h, suggest- ing a secondary signaling event due to new protein synthesis or autocrine signaling. Consistent with increased mRNA levels, the enhancing effect of rapamycin on IRF-1 protein induction VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 8 JOURNAL OF BIOLOGICAL CHEMISTRY 24348 VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 Regulation of STAT1 by mTOR FIGURE 5. Inhibition of mTOR, or depletion of 4/PP2Ac, augments STAT1 and IRF-1 expression in response to IFN-. A549 cells were incubated with serum-free medium in the absence or presence of rapa- mycin, 50 ng/ml, for 1 h before addition of IFN-, 100 units/ml, for 0.5, 6, or 18 h before detection of IRF-1 or STAT1 mRNA (A) or 0.5, 1, or 2 h before detection of IRF-1 protein (B). In panels C–E, cells were transfected with control or siRNA targeting mTOR, 4, or PP2Ac for 72 h before serum withdrawal for 1 h in the absence or presence of rapamycin, incubation without or with IFN- for 2 h, and detection of the indicated proteins by Western blot. Average integrated band density for IRF-1 S.E. (, p 0.05 rapamycin with IFN- versus IFN-) is shown below each Western blot. F, similarly siRNA-transfected cells were exposed to IFN- in the absence or presence of rapamycin for 18 h before detection of STAT1 mRNA levels by real-time PCR. Changes in mRNA levels are expressed as -fold change relative to control mRNA levels 1 (Ct method, means of triplicate samples S.E.), and are representative of three experiments (, p 0.05 versus IFN- alone in control-trans- fected cells). Gels in panels B–E represent data from 3–4 independent experiments. dependent genes. We further pro- posed that this effect of mTOR should be independent of riboso- mal biogenesis, which in yeast is Tap42 (4)-independent. The human inducible nitric oxide syn- thase (hiNOS), Fas (CD95), and caspase-1 genes encode pro-in- flammatory and pro-apoptotic proteins that play essential roles in the innate immune response and the control of cell death. All three are induced by IFN- in STAT1- and IRF-1-dependent fashion (22, 26, 27). Consistent with its enhancing effect on IRF-1 and STAT1 (Fig. 5), rapamycin aug- mented subsequent hiNOS, Fas, and caspase-1 mRNA levels by 25–38% at 18 h after exposure to IFN- (Fig. 6A). There was no sig- nificant effect of rapamycin on late IFN--stimulated gene expression at 6 or 12 h. Although this is con- sistent with a requirement for de novo IRF-1 and STAT1 synthesis, a STAT1-independent effect of rapamycin, or an autocrine mech- anism, could not be ruled out. Unlike STAT1-dependent genes, inhibition of mTOR did not aug- ment levels of precursor ribosomal RNA (i.e. 45 S rRNA). Regulation of STAT1 by mTOR In panels C–E, cells were transfected with control or siRNA targeting mTOR, 4, or PP2Ac for 72 h before serum withdrawal for 1 h in the absence or presence of rapamycin, incubation without or with IFN- for 2 h, and detection of the indicated proteins by Western blot. Average integrated band density for IRF-1 S.E. (, p 0.05 rapamycin with IFN- versus IFN-) is shown below each Western blot. F, similarly siRNA-transfected cells were exposed to IFN- in the absence or presence of rapamycin for 18 h before detection of STAT1 mRNA levels by real-time PCR. Changes in mRNA levels are expressed as -fold change relative to control mRNA levels 1 (Ct method, means of triplicate samples S.E.), and are representative of three experiments (, p 0.05 versus IFN- alone in control-trans- fected cells). Gels in panels B–E represent data from 3–4 independent experiments. ing effect of rapamycin on IRF-1 and STAT1 induction. The effect of rapamycin on STAT1 expression is sustained, and follows the de novo synthesis of IRF-1. Inhibition of mTOR Enhances the Induction of Late IFN-- stimulated Genes—Given the sustained increase in STAT1 and IRF-1 levels upon inactivation of mTOR or its associated phosphatases, we hypothesized that mTOR, PP2Ac, and 4 also control the induction of late IFN--stimulated STAT1- To confirm that the effect of rapamycin reflected a requirement for mTOR activity, we used molecular mTOR inhibitors and measured caspase-1 induction. siRNA-medi- ated depletion of mTOR (Fig. 7, A and B), as well as the expression of kinase-dead recombinant mTOR (Fig. 7C), sig- SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 JOURNAL OF BIOLOGICAL CHEMISTRY 24349 SEPTEMBER 4, 2009•VOLUME 284•NUMBER 36 24349 Regulation of STAT1 by mTOR FIGURE 6. Inhibition of mTOR, 4, or PP2A augments the induction of STAT1- and IRF-1-dependent IFN--stimulated genes. A549 cells were incubate with serum-free medium in the absence or presence of rapamycin, 50 ng/ml, before addition of IFN-, 100 units/ml, for 6, 12, or 18 h. Changes in caspase-1, Fa and hiNOS mRNA (, p 0.05 rapamycin with IFN- versus IFN- alone) (A), or 45 S rRNA levels (, p 0.05 versus control) (B) are expressed as -fold increas relative to control 1 (Ct method, means of triplicate samples S.E.), and represent data from three experiments. DISCUSSION nificantly enhanced the induction of caspase-1 mRNA and protein levels at 18 h. As expected, phosphorylation of S6K or Akt was reduced in unstimulated mTOR-deficient cells (Fig. 7, B and C, lane 5 versus 1). In contrast to the 2-h time point (Fig. 5C), phosphorylation of S6K or Akt in IFN-- stimulated cells was intact, perhaps due to preserved endog- enous mTOR activity, or the contribution of other signaling pathways. As was the case for IRF-1 (Fig. 5), caspase-1 levels were enhanced by rapamycin regardless of Akt or S6K phos- phorylation state (Fig. 7, B and C, lane 6 versus lanes 4 and 8). Depletion of mTOR or expression of kinase-dead mTOR (Fig. 7, A–C) blocked the enhancing effect of rapamycin on caspase-1 induction, indicating that the effect of rapamycin occurred via mTOR. In this study, we identified a novel mechanism by which mTOR controls the transcription factor STAT1. mTOR and the phosphatase subunits 4 and PP2Ac associate with STAT1 in a dynamic protein complex. Inactivation of mTOR favors its association with STAT1 and promotes both constitutive and IFN--induced STAT1 nuclear content. Inactivation of mTOR, 4, or PP2Ac enhances the expression of STAT1-dependent genes and proteins involved in inflammation and apoptosis. The effect of mTOR on STAT1 does not correlate with the phosphorylation status of other known mTOR cell growth effectors (e.g. S6K and Akt) (Figs. 5–7). The regulation of STAT1 by mTOR and 4 is reminiscent of TOR/Tap42-de- pendent control of Msn2 in yeast (5) and suggests an evolution- arily conserved mechanism by which mTOR controls cellular stress responses. To determine whether caspase-1 induction by IFN- is reg- ulated by growth factors, and whether mTOR is required, A549 cells were incubated without or with serum in the absence or presence of rapamycin. The induction of caspase-1 protein lev- els by IFN- was enhanced by rapamycin (Fig. 7D, lane 6 versus 2). Incubation of cells with serum suppressed the induction of caspase-1 by IFN- (lane 4 versus 2), and this effect was reversed by the addition of rapamycin (lane 8 versus 4), indicat- ing a role for mTOR in dampening IFN--stimulated gene and protein induction by growth factors. We show that inactivation of mTOR amplifies the induction of early (i.e. IRF-1) and late (i.e. hiNOS, caspase-1, and Fas) IFN--stimulated genes. Others have shown that early induc- tion of IRF-1 requires rapid translocation of activated STAT1 to the nucleus (29). Regulation of STAT1 by mTOR C, STAT1-deficient (U3A) or contro (2fTGH) transformed human fibroblasts were exposed to serum-free medium in the absence or presence of rapamycin before addition of IFN- for 0 or 18 h an measurement of 45 S rRNA levels (, p 0.05 versus control). D, A549 cells were transfected with siRNAs targeting 4 or PP2Ac for 72 h before serum deprivationintheabsenceorpresenceofrapamycinandincubationwithIFN-for18h.ChangesinhiNOSorcaspase-1mRNAlevelsarerepresentativeofthre individual experiments. , p 0.05 versus IFN- alone in control-transfected cells. IGURE 6. Inhibition of mTOR, 4, or PP2A augments the induction of STAT1- and IRF-1-dependent IFN--stimulat FIGURE 6. Inhibition of mTOR, 4, or PP2A augments the induction of STAT1- and IRF-1- FIGURE 6. Inhibition of mTOR, 4, or PP2A augments the induction of STAT1- and IRF-1-dependent IFN--stimulated genes. A549 cells were incubated with serum-free medium in the absence or presence of rapamycin, 50 ng/ml, before addition of IFN-, 100 units/ml, for 6, 12, or 18 h. Changes in caspase-1, Fas, and hiNOS mRNA (, p 0.05 rapamycin with IFN- versus IFN- alone) (A), or 45 S rRNA levels (, p 0.05 versus control) (B) are expressed as -fold increase relative to control 1 (Ct method, means of triplicate samples S.E.), and represent data from three experiments. C, STAT1-deficient (U3A) or control (2fTGH) transformed human fibroblasts were exposed to serum-free medium in the absence or presence of rapamycin before addition of IFN- for 0 or 18 h and measurement of 45 S rRNA levels (, p 0.05 versus control). D, A549 cells were transfected with siRNAs targeting 4 or PP2Ac for 72 h before serum deprivationintheabsenceorpresenceofrapamycinandincubationwithIFN-for18h.ChangesinhiNOSorcaspase-1mRNAlevelsarerepresentativeofthree individual experiments. , p 0.05 versus IFN- alone in control-transfected cells. Regulation of STAT1 by mTOR FIGURE 7 TOR ki ti it i i d f th i is, rapamycin enhanced IRF-1 mRNA at 0.5 but not at 6 h. Similarly, the effect of rapamycin on late IFN--stimulated genes (i.e. 12 and 18 h) followed the new synthesis of IRF-1 and STAT1. Although we cannot exclude a STAT1-independent effect of rapamycin on late IFN--stimulated genes, the enhancement of their mRNA and protein levels coincided with elevated nuclear STAT1 levels (Fig. 2, A and B) and required mTOR, 4, and PP2Ac (Figs. 6 and 7). g The enhancing effect of mTOR inactivation on STAT1 exhibits several similarities to the TOR/Msn2-mediated “stress response” in S. cerevisiae (5). As was the case for TOR and Msn2 (30), we could not detect a role for mTOR in the phosphoryla- tion of STAT1 (6) (Figs. 2 and 5C, and supplemental Fig. S1). Furthermore, rapamycin increased the physical association between mTOR and STAT1 (Fig. 1D, rapamycin alone), as well as STAT1 nuclear content (Figs. 2 (lane 3), 3, and 4), in the absence if IFN- (i.e. under conditions where STAT1 was not phosphorylated at Ser-727 or Tyr-701). Therefore, mTOR reg- ulates the constitutive trafficking of STAT1 via a rapamycin- sensitive mechanism that involves an enhanced association between inactive mTOR and STAT1. As is the case for yeast TOR/Msn2, the precise molecular target for regulation of STAT1 nuclear transport by mTOR kinase or PP2Ac phospha- tase activity is unknown and awaits further phospho-proteomic characterization of the complex and its components. In contrast to its stimulation of protein synthesis and cell growth, mTOR attenuated the induction of STAT1-dependent genes. Yeast TOR and Tap42 similarly modify stress response genes by reducing the amount of Msn2 in the nucleus. In con- trast to Msn2- or STAT1-dependent stress-induced genes, TOR can also control constitutive levels of nutrient-sensitive genes (5, 31). In yeast, “nutrient discrimination pathway” genes are regulated by a different transcription factor (i.e. Gln3) and its scaffolding protein Ure2. Unlike Msn2 or STAT1, however, rapamycin blocked the phosphorylation of Ure2 and Gln3 (32). As was the case for yeast Msn2 or Gln3, mTOR control of STAT1 was functionally and genetically distinct from its effect on the synthesis of ribosomal RNA precursors (Fig. 6, B and C). DISCUSSION Newly synthesized IRF-1 permits additional synthesis of STAT1 (10). The increased IRF-1 and STAT1 con- tribute to the induction of late IFN--stimulated genes, which VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 24350 JOURNAL OF BIOLOGICAL CHEMISTRY 24350 VOLUME 284•NUMBER 36•SEPTEMBER 4, 2009 Regulation of STAT1 by mTOR Our experiments in 4-depleted cells, or those expressing con- stitutively active or dominant-negative PP2Ac mutants, place 4 and PP2Ac downstream of mTOR in its control of STAT1, and STAT1-dependent genes (Figs. 4–7). These results impli- cate mTOR, its associated phosphatases, and STAT1 in a par- tially conserved mechanism that regulates the levels of stress- induced genes. FIGURE 7. mTOR kinase activity is required for the suppression of Caspase-1 induction. A549 cells were transfected with control or siRNA tar- i TOR f 72 h b f i hd l f 1 h i h b FIGURE 7. mTOR kinase activity is required for the suppression of Caspase-1 induction. A549 cells were transfected with control or siRNA tar- geting mTOR for 72 h before serum withdrawal for 1 h in the absence or presence of rapamycin, incubation without or with IFN- for 18 h, and detec- tion of caspase-1 mRNA levels by real-time PCR (A) or caspase-1 protein levels by Western blot (B). C, Myc-tagged wild-type (WT) or kinase-dead (KD) mTOR were expressed in A549 cells, before incubation without or with rapamycin, 50 ng/ml, in serum-free medium for 1 h followed by the addition of IFN- for 18 h. D, A549 cells were exposed to IFN- in the absence or presence of rapa- mycin or serum for 18 h, before preparation of whole cell lysates and detec- tion of caspase-1 by Western blot analysis. Changes in mRNA levels (A) are expressed as -fold change relative to control mRNA levels 1 (Ct method, means of triplicate samples S.E.), and are representative of three experi- ments (, p 0.05 versus IFN- alone in control-transfected cells). Protein levels (mean integrated band density for nuclear STAT1 S.E.) were meas- uredbydensitometryandareshowntotherightofeachWesternblot.Results from Western blots (B–D) are representative of 3–4 individual experiments (, p 0.05 versus IFN- alone). FIGURE 7. mTOR kinase activity is required for the suppression of Caspase-1 induction. A549 cells were transfected with control or siRNA tar- geting mTOR for 72 h before serum withdrawal for 1 h in the absence or presence of rapamycin, incubation without or with IFN- for 18 h, and detec- tion of caspase-1 mRNA levels by real-time PCR (A) or caspase-1 protein levels by Western blot (B). Regulation of STAT1 by mTOR (2004) Nat. Cell Biol. 6, 358–365 The role of mTOR in cell growth, proliferation, or death, is a principal preoccupation in research on clinical proliferative disorders, such as tuberous sclerosis complex and lymphangio- leiomyomatosis, which involve germ line or mosaic loss of the endogenous mTOR suppressor tuberin (43). However, in ani- mal models and clinical trials, rapamycin, which is intended to block cell growth and proliferation, only partially inhibits tumor or disease progression (44, 45), suggesting that alterna- tive signaling mechanisms might be targeted. In fact, tuberous sclerosis complex 2-deficient tumors are particularly sensitive to the growth inhibitory effects of IFN- and exhibit dysregu- lated STAT1 signaling (46). Consistent with these in vivo obser- vations, we show that, when mTOR activity is reduced, assem- bly of the mTORSTAT1 complex can enhance STAT1 nuclear content and the induction of genes involved in apoptosis inde- pendently of other known mTOR effectors, such as S6K, Akt, or 4E-BP1. Targeting the physical association between mTOR and STAT1 might therefore represent an additional opportunity to amplify tumor suppressor activity (e.g. IRF-1 and STAT1) and curb the progression of clinical conditions that exhibit exces- sive mTOR activity. 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https://openalex.org/W2152916845	https://europepmc.org/articles/pmc3869860?pdf=render	English	null	Neural Correlates of Self-Appraisals in the Near and Distant Future: An Event-Related Potential Study	PloS one	2,013	cc-by	6,157	Introduction Mental time travel is one of the most fascinating characteristics of the human mind [1–3]. Mental time travel results in temporal selves; that is, people construct their self-identity by recalling their past and imagining a hypothetical future self [4,5]. Moreover, perceptions of temporal selves can enhance self-regulation abilities and activate goal-related behaviors. For example, simulations of hypothetical future selves helped middle school students increase their school involvements and adaptive behaviors [6]. Perceptions of temporal selves can also influence decisions. For instance, people who perceive and treat the future self differently from the present self are prone to making short-sighted decisions [7]. Therefore, it is important to understand how people construct and represent their temporal selves. In this study, we focused on people’s perception of the future self. Temporal distance influences perceptions of the future self. Although people view the future self favorably, the degree of favorability is affected by temporal distance. Construal level theory has been employed to explain mechanisms underlying the effect of temporal distance on future self-appraisals [16]. From this perspective, whereas the near future self is associated with a low- level, concrete construal, the distant future self is associated with a high-level, abstract construal [17–19]. To elaborate, the near future self-construal is more grounded in concrete events and comprises complex representations of the self that include both positive and negative self-construals. In contrast, because the distant future self is relatively remote from direct life experiences and people have a tendency to view the future in a positive light [8,10,20,21], distant future self-views should be even more favorable and positive. In support of construal level theory, Heller et al. (2011) found perceptions of (1) affect, (2) traits, and (3) narratives of one’s distant future self (i.e., three years from now) were more positive and less variable than perceptions of one’s near future self (i.e., a month from now) in three independent studies [16]. Similarly, Kanten and Teigen (2008) found that people predicted having a more favorable future self in two years’ as opposed to six months’ time [11]. There is considerable evidence that people’s future self is dominated by favorable self-views. People believe that they are more likely to experience positive events and less likely to experience negative events compared to their peers [8,9]. Abstract To investigate perceptual and neural correlates of future self-appraisals as a function of temporal distance, event-related potentials (ERPs) were recorded while participants (11 women, eight men) made judgments about the applicability of trait adjectives to their near future selves (i.e., one month from now) and their distant future selves (i.e., three years from now). Behavioral results indicated people used fewer positive adjectives, more negative adjectives, recalled more specific events coming to mind and felt more psychologically connected to the near future self than the distant future self. Electrophysiological results demonstrated that negative trait adjectives elicited more positive ERP deflections than did positive trait adjectives in the interval between 550 and 800 ms (late positive component) within the near future self condition. However, within the same interval, there were no significant differences between negative and positive traits adjectives in the distant future self condition. The results suggest that negative emotional processing in future self- appraisals is modulated by temporal distance, consistent with predictions of construal level theory. Received March 21, 2013; Accepted November 22, 2013; Published December 20, 2013 Received March 21, 2013; Accepted November 22, 2013; Published December 20, 2013 Copyright: 2013 Luo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by ‘‘the Fundamental Research Funds for the Central Universities’’ (SWU1209469) to the first author, the Key Discipline Fund of the National 211 Project, China (NSKD11001), and the Research Team’s Construction Project from the Faculty of Psychology in Southwest University (2012) "The Mechanism and Application of Range-Synthesis Model of Time’’ (TR201201-1) to the corresponding author and research fellow. Study design, data collection and analysis were supported by The Fundamental Research Funds for the Central Universities (SWU1209469) and the Key Discipline Fund of the National 211 Project, China (NSKD11001). The Research Team’s Construction Project from the Faculty of Psychology in Southwest University (2012) "The Mechanism and Application of Range-Synthesis Model of Time’’ (TR201201-1) has roles in preparation of the manuscript and, decision to publish. Competing Interests: The authors have declared that no competing interests exist. Competing Interests: The authors have declared that no competing interests exist. * E-mail: xthuang@swu.edu.cn * E-mail: xthuang@swu.edu.cn pessimistic view to the future is related to depressive disorders [14,15]. Neural Correlates of Self-Appraisals in the Near and Distant Future: An Event-Related Potential Study Yangmei Luo1,2, Todd Jackson1,2, Xiaogang Wang1,2, Xiting Huang1,2* 1 Key Laboratory of Cognition and Personality (SWU), China Ministry of Education, Chongqing, China, 2 Faculty of Psychology, Sou Yangmei Luo1,2, Todd Jackson1,2, Xiaogang Wang1,2, Xiting Huang1,2* 1 Key Laboratory of Cognition and Personality (SWU), China Ministry of Education, Chongqing, China, 2 Faculty of Psychology, Southwest University, Chongqing, China December 2013 \| Volume 8 \| Issue 12 \| e84332 Procedure Participants made self-judgments on the same set of adjectives in two conditions. Specifically, they were instructed to decide whether or not the 80 adjectives described characteristics they might have one month from now (Near Future Self), and characteristics they might have three years from now (Distant Future Self). Each adjective was presented twice within the two self conditions (i.e., 160 trials per condition). Within each condition, there were four blocks of 40 trials each. The interval between trials was 1000–1500 ms. Conditions and blocks were presented in random order. To prime the corresponding temporal self, participants were asked to describe in writing their image of the corresponding temporal self for two minutes before initiating each condition [5]. Subsequently, all trials for that self-judgment condition followed the priming task. First, a fixation point appeared for 500–750 ms in the center of the screen and was followed by a cue for the temporal self condition presented for 250 ms (‘‘the self one month later’’ ‘‘the self three years from now’’). After a 400–800 ms interval, a trait adjective was presented for 3000 ms. Participants were asked to respond as accurately and quickly as possible regarding the extent to which each adjective described their near (or distant) future self on a 4-point rating scale (very unsuitable = 1, unsuitable = 2, suitable = 3, very suitable = 4). Based on Moran et al. [38], responses of 1 or 2 were considered low in self-relevance and responses of 3 or 4 were considered high in self-relevance. Drawing upon the methodology and hypotheses from Heller et al.’s [17] research as well as contentions of construal level theory that the near future self-construal is relatively concrete and comprised of complex representations that include both positive and negative self-conceptions while the distant future self is more abstract and idealized in a more uniformly positive way [16–19], we assessed interactions between temporal distance and valence on behavioral and electrophysiological data. Specifically, it was hypothesized that people would have more favorable distant future self-view than near future self-view. Moreover, negative adjectives for the near future self should elicit a larger amplitude of LPC than positive adjectives, while emotional adjective should have no effect within the distant future self condition. To assess these hypotheses, participants evaluated their near and distant After the ERP procedure, more information was collected on participant performance during the task. Ethics Statement The School of Psychology Research Ethics Committee of Southwest University (SWU) granted ethical approval for the study and all participants gave written informed consent to the experimental procedure. They were informed that they had the right to withdraw at any time during the experiment. In light of mixed findings, further research is warranted to evaluate the extent to which attributes of the near or distant future self are perceived as more favorable. Furthermore, much of the associated research has focused on perceived behavioral differ- ences between near and distant future self-appraisal [11,16,23], so it is less clear whether self-appraisals of near and remote future distant selves correspond to distinct underlying neural responses. Evaluation of neural correlates related to evaluating attributes of near and distant future selves can elucidate how temporal distance is related stage of processing self-appraisals, because such judgments may occur within a few hundred milliseconds, well before behavioral responses. With high time resolution on the order of milliseconds, ERPs provide an excellent means to evaluate the time course of future self-appraisal processing as a function of temporal distance. Stimuli The 40 positive and 40 negative trait adjectives used in the current study were the same as those used in previous research [27]. Mean valences were 5.61 (SE = 0.04) for positive traits adjectives and 2.72 (SE = 0.07), for negative traits adjectives using a 7-point rating scale anchored at 1 (least desirable) and 7 (most desirable). Positive and negative trait adjectives were matched for familiarity, meaningfulness, and complexity (i.e., number of strokes needed to write the Chinese character). In relation to components of ERP, the late positive component (LPC) occurs between 300 and 1200 ms after stimulus onset [24], and is theorized to indicate emotional processing. The amplitude of LPC is usually larger for both positive and negative than for neutral stimuli [25,26]. However, for direct comparisons of LPC amplitudes elicited by positive and negative stimuli, results have been mixed. Some studies have found negative stimuli elicit larger LPC [27–29] than positive stimuli do, while the reverse pattern [30,31] or null effects [25] have also been observed. Despite such discrepancies, the positivity of LPC to emotional stimuli is associated with later controlled cognitive evaluation of emotional meanings of stimuli [26,29,32,33]. Furthermore, LPC effects have been implicated in episodic memory retrieval and mental imagery [34–36]. For example, Rugg et al. (1998) found LPC amplitude was larger for more deeply encoded items [34]. Also, Kanske & Kotz (2007) found that concrete words, which are prone to mental imagery, evoked an LPC effect while there was no effect on abstract words [37]. In another relevant study, negative words elicited a larger positive LPC deflection relative to positive words when evaluating present and past selves. However, in the same interval, there was no a significant LPC effect when evaluating the future selves. It was speculated that the negative words could evoke more specific episodic events in the past and present selves relative to future selves [28]. Participants ERP recordings were obtained from 19 undergraduates (eleven women, eight men) aged 19–22 years (mean age, 21.01 years) from SWU in Chongqing, China. ERP data from one participant was removed due to excessive artifacts. All participants engaged in the experiment as paid volunteers, had no history of current or past neurological or psychiatric illness, and had normal or corrected-to- normal vision. Self-Appraisals in the Near and Distant Future people might expect better outcomes for the distant future self and note remote future selves are unlikely to be disparaged [4]. At the same time, however, people ‘‘may simply be motivated to exaggerate the glories of psychologically proximal future selves more than those of distant future selves’’ (Peetz & Wilson, 2008: 2097) [4], to maintain favorable current self-regard [4,22,23]. In support of these ideas, Wilson et al. (2012) manipulated subjective temporal distance of a future self by holding objective or calendar time constant, and found participants predicted having more favorable personal attributes at a close future time than a distant future time [23]. future selves across a series of positive and negative trait adjectives within an ERPs experiment. Following Heller et al., [16], the near future self were operationalized as the self in one month’s time, and the distant future self was operationalized as the self in three years’ time. December 2013 \| Volume 8 \| Issue 12 \| e84332 Introduction Positive future events are also generated more easily and quickly than negative future events [10] and people evaluate their future selves as having more desirable traits than their present and past selves [11]. Notably, the dominance of favorable self-views for the future self has important implications for mental health. Specifically, perceptions of a moderately desirable future may promote motivation, mental health and well-being [12,13]. In contrast, a However, research on perceptions of near versus distant future selves is not uniformly consistent. Theorists using temporal self- appraisal theory have also identified circumstances in which December 2013 \| Volume 8 \| Issue 12 \| e84332 PLOS ONE \| www.plosone.org 1 December 2013 \| Volume 8 \| Issue 12 \| e84332 Self-Appraisals in the Near and Distant Future Event-related potential recording and analysis Continuous brain electrical activity was recorded from 64 scalp sites using tin electrodes mounted in an elastic cap (Brain Product, Munchen, Germany), with the reference on left and right mastoids. The vertical electrooculogram (EOG) was recorded with electrodes placed above and below the left eye. All inter- electrode impedance was maintained below 5 kV. The electroen- cephalogram (EEG) and EOG were amplified using a DC-100Hz bandpass and continuously sampled at 500 Hz/channel for off- line analysis. Eye movement artifacts were corrected with the Gratton–Coles algorithm using the EOG data [23]. After this, trials with EOG artifacts (mean EOG voltage exceeding 6100 mV) and those contaminated with artifacts due to amplifier clipping, bursts of electromyographic (EMG) activity, or peak-to-peak deflection exceeding 6100 mV were excluded from averaging. Following from other published studies [40,41], a 16 Hz low pass filter was used. A repeated-measures ANOVA (2: near, distant x 2: positive, negative) on reaction times found a main effect for valence (F (1, 18) = 16.23, p,.001, n2 p = .47) (Table 1). RTs for positive traits (M = 890.12, SE = 39.00) were significantly faster than RTs for negative traits (M = 940.36, SE = 41.39) (t(18) = 24.03, p = .001, Cohen’s d = 2.29). However, the main effect for time (F (1, 18) = .78, p = .387, n2 p = .04) and temporal distance x valence interaction (F (1, 18) = 3.01, p = .100, n2 p = .14) were not significant. On self-report measures assessed following the ERPs session, paired samples t-tests showed that participants evaluated their present selves as more connected with their near future selves (M = 5.79, SE = .36) than their distant future selves (M = 4.47, SE = .29) (t(18) = 3.51, p = .003, Cohen’s d = .93). Finally, as expected, specific events were more likely to come to mind when evaluating the near future self (M = 5.05, SE = .30) than the distant future self (M = 4.16, SE = .34) (t(18) = 2.30, p = .034, Cohen’s d = .63). A repeated-measures ANOVA (2: near, distant x 2: positive, negative) on reaction times found a main effect for valence (F (1, 18) = 16.23, p,.001, n2 p = .47) (Table 1). Self-Appraisals in the Near and Distant Future positive and negative traits in each judgment condition are shown in Table 1. A repeated-measures ANOVA (2: near, distant x 2: positive, negative) found a main effect of valence (F (1, 18) = 319.75, p,.001, n2 p = .95) that was qualified by an interaction between temporal distance and valence (F (1, 18) = 12.30, p = .003, n2 p = .41). No significant main effect of temporal distance was found (F (1, 18) = .09, p = .925, n2 p = .00). A simple effects analysis of temporal distance x valence interaction showed that ‘‘high’’ responses for positive trait adjectives in the near future self condition (M = 87.83%, SE = 2.67%) were significantly lower than those in the distant future self condition (M = 92.43%, SE = 1.85%) (t(18) = 22.64, p = .017, Cohen’s d = 2.46). Negative trait adjec- tives showed the opposite pattern: ‘‘high’’ responses in the near future self condition (M = 14.67%, SE = 3.38%) were significantly higher than in the distant future self condition (M = 10.26%, SE = 2.86%) (t(18) = 2.85, p = .011, Cohen’s d = .32). (using a 7-point rating scale: 1 = not at all, 7 = very frequently). In addition, participants completed a modified version of the Future Self-Continuity Scale [39]. This scale assessed the connectivity between the current self and each type of future self with depictions of two circles that ranged from no overlap to almost complete overlap. Participants selected the circle pair that best described how connected they felt to a future self one month later or three years from now. Event-related brain potential waveforms analysis As shown in Figure 1, the N1 (50–150 ms), P2 (150–300 ms), N2 (300–400 ms) and late positive component (LPC) were elicited by both near and distant future self conditions. y Main effects for trait valence and temporal distance were not significant for N1, P2 or N2. From ERP waveforms, we found that, relative to positive traits, negative traits for the near future self elicited a more positive ERP deflection than for distant future selves in the interval between 550 ms and 800 ms(LPC). A repeated-measures ANOVA (2: near, distant x 2: positive, negative x 9: electrode site) predicting the amplitude of LPC found a marginal effect for valence (F (1, 17) = 4.25, p = .055, n2 p = .20) and a significant temporal distance x trait valence interaction (F (1, 17) = 5.20, p = .036, n2 p = .23). None of the other comparisons were significant (all ps..05). Mean LPC amplitudes elicited by negative trait adjectives (M = 3.22, SE = 1.31) were more positive than those elicited by positive trait adjectives (M = 1.66, SE = .99) when evaluating the near future self (t(17) = 2.78, p = .013, Cohen’s d = .32). There was no such difference between negative traits (M = 2.85, SE = 1.02) and positive traits (M = 2.40, SE = .87) in the distant future self condition (t(17) = .85, p = .406, Cohen’s d = .11). As shown in Figure 1, these differences were evident in the central- parietal cortex. Neither main effects nor interactions for mean LPC amplitudes were significant between 400–550 ms and 800– 1000 ms. Event-related potential recording and analysis RTs for positive traits (M = 890.12, SE = 39.00) were significantly faster than RTs for negative traits (M = 940.36, SE = 41.39) (t(18) = 24.03, p = .001, Cohen’s d = 2.29). However, the main effect for time (F (1, 18) = .78, p = .387, n2 p = .04) and temporal distance x valence interaction (F (1, 18) = 3.01, p = .100, n2 p = .14) were not significant. On self-report measures assessed following the ERPs session, paired samples t-tests showed that participants evaluated their present selves as more connected with their near future selves (M = 5.79, SE = .36) than their distant future selves (M = 4.47, SE = .29) (t(18) = 3.51, p = .003, Cohen’s d = .93). Finally, as expected, specific events were more likely to come to mind when evaluating the near future self (M = 5.05, SE = .30) than the distant future self (M = 4.16, SE = .34) (t(18) = 2.30, p = .034, Cohen’s d = .63). Respective EEG averages for the four conditions of interest (positive-near future self, negative-near future self, positive-distant future self, and negative distant future self) were calculated. The averaged epoch for ERPs was 1200 ms including a 200 ms pre- response baseline. Electrodes of interest were analyzed with a repeated-measures analysis of variance (ANOVA) (2: Near, Distant x 2: Positive, Negative x 9: Fcz, Fc3, Fc4, Cz, C3, C4, CPz, CP3, CP4). When a main effect was found, a Bonferroni- corrected post-hoc t-test for multiple comparisons was used to determine the significance of the difference for each pair-wise comparison. For all analyses, p values were corrected for deviations according to the Greenhouse–Geisser method. Procedure Specifically, participants were asked to rate the frequency with which specific events came to their mind when evaluating their near and distant future selves December 2013 \| Volume 8 \| Issue 12 \| e84332 December 2013 \| Volume 8 \| Issue 12 \| e84332 PLOS ONE \| www.plosone.org 2 Self-Appraisals in the Near and Distant Future Behavioral performance Judgments of each future self condition were collapsed into high (3 and 4 responses) and low (1 and 2 responses) self-relevance categories. Mean proportions of ‘‘high’’ responses given for Table 1. Behavioral measures as a function of future self condition. Near future self Distant future self Proportion of ‘‘high’’ responses (%) Positive traits 87.83(2.67) 92.43(1.85) Negative traits 14.67 (3.38) 10.26(2.86) Response times (ms) Positive traits 917.97(42.58) 907.98(40.34) Negative traits 953.46(39.72) 976.28(45.74) Connectivity with future self 5.79(.36) 4.47(.29) Frequency of specific events 5.05(.30) 4.16(.34) Note: standard errors are shown in parentheses doi:10.1371/journal.pone.0084332.t001 Table 1. Behavioral measures as a function of future self condition. Table 1. Behavioral measures as a function of future self condition. Pearson correlations were run to explore whether LPC amplitudes (550 ms and 800 ms) correlated with reported psychological connectivity and event recall frequency when December 2013 \| Volume 8 \| Issue 12 \| e84332 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 3 Self-Appraisals in the Near and Distant Future Figure 1. Grand average event-related brain potentials and topographical maps for the difference wave. A: Grand average event- related brain potentials. ERPs evoked by positive and negative traits for the near and distant future condition, and the difference wave (negative near future condition-positive near future condition) at FC3 and Cz. B: topographical maps of the voltage amplitudes for the difference wave at 686–712 and 714–740 ms. doi:10.1371/journal.pone.0084332.g001 Figure 1. Grand average event-related brain potentials and topographical maps for the difference wave. A: Grand average event- related brain potentials. ERPs evoked by positive and negative traits for the near and distant future condition, and the difference wave (negative near future condition-positive near future condition) at FC3 and Cz. B: topographical maps of the voltage amplitudes for the difference wave at 686–712 and 714–740 ms. doi:10.1371/journal.pone.0084332.g001 participants evaluated their future selves. No significant correla- tions were found (see Table 2), but the highest correlation for ERP amplitude was for frequency of specific events recalled in the near negative future self condition (r = .317, p = 0.2) relative to other conditions. to their distant future selves (i.e. three years from now). Moreover, while making judgments, participants reported specific events came to mind more often and they felt more psychologically connected to the near future self rather than the distant future self. Behavioral performance Behavioral results about future personality attributes were consistent with select previous studies indicating people have more favorable distant future self-appraisals than near future self- appraisals [11,16]. These findings demonstrated how people’s predictions of their future personality might depend on how far they project into the future, with a more highly optimistic bias regarding the distant future self, at least within this experimental paradigm. December 2013 \| Volume 8 \| Issue 12 \| e84332 Self-Appraisals in the Near and Distant Future Electrophysiological results indicated ERP components (i.e., N1, P2 and N2) associated with early stages of visual and semantic processing did not differ between near and distant future self conditions. However, in the current study, temporal distance significantly interacted with emotional valence to predict LPC. For the near future self, larger LPC amplitudes were elicited by negative trait adjectives relative to positive trait adjectives from 550 ms to 800 ms after stimulus onset over the central-parietal region. In contrast, the distant future self did not show a significant difference in LPC elicited by negative versus positive traits within this interval. appraisals, consistent with construal level theory. These findings may also support previous research showing how level of abstraction can interact with emotional valence. Specifically, Kanske & Kotz (2007) found that concrete negative words (low level construal) elicited larger LPC amplitude than concrete neutral or concrete positive words while there was no emotional effect on abstract words [37]. Although the results provided further evidence that temporal distance modulated future self-appraisals, it is worth noting limitations of the present study and specific directions for future work. First, effect sizes were relatively small, perhaps because people had relatively favorable future self-views in both future self conditions. On a related note, it is not clear how well findings generalize beyond the current methodology, given that other researchers have also found replicable effects of a more favorable near future self using different research measures and designs [23]. Future research is needed to clarify the robustness of effects across study paradigms including those that feature manipulations of subjective time rather than calendar time [23]. Third, following previous studies [33], it would be useful to separate self-referential processing from emotional valences of trait adjectives in future work. Fourth, based upon thoughtful suggestions from a reviewer, optimal experimental designs for future study should include present self-appraisals as a control condition that would permit the examination of temporal distance on self-appraisals in a more precise way. Finally, a deeper understanding of these processes may result from extensions to depressed or anxiety disordered participants, given that these groups tend to have relatively more pessimistic views of their future selves (e.g. [42]). In emotional electrophysiological studies, it is well established that the LPC reflects the elaborate and controlled late processing of emotional stimuli. 3. D’Argembeau A, Xue G, Lu Z-LL, Van der Linden M, Bechara A, et al. (2008) Neural correlates of envisioning emotional events in the near and far future. Neuroimage 40: 398–407. Acknowledgments The authors would like to thank Dr. Jennifer Beer from University of Texas at Austin for her proofreading and valuable comments on the manuscript. They are also grateful to three anonymous reviewers and the editor for their helpful comments. Conclusions In sum, our behavioral and electrophysiological results were consistent with central assumptions of construal level theory. From this perspective, the near future self is related to a complex, low level, concrete construal characterized by positive and negative attributes while the distant future self is related to a high level, abstract construal characterized by idealized, schematic thinking [17]. The near future self construal is relatively more grounded in mixed valence experiences of daily life compared with the more highly idealized distant future self construal. Findings that participants used relatively fewer positive adjectives and more negative adjectives, recalled more specific events and perceived relatively increased connectedness in the near future self condition were in line with assumptions of the construal approach. In summary, the present study demonstrated that neural substrates of future self-appraisals were modulated by temporal distance. The results showed that, when reflecting on the near future self, negative trait adjectives elicited more positive ERP deflections than positive trait adjectives in the interval between 550 and 800 ms (LPC). Conversely, there were no significant differences in ERP deflections elicited by negative and positive traits adjectives when evaluating the distant future self in the same interval. The findings suggest temporal distance modulates negative emotional processing in future self-appraisals, which is predicted by construal level theory. Future research is needed to understand how these results generalize to changes in subjective time and populations that veer toward pessimistic future self- appraisals. Nonetheless, these results were also partially consistent with temporal self-appraisal theory [4,22,23] which posits people show optimistic biases towards both temporal selves due, in part, to motivation to protect current self-regard (self-enhancement) [4,22]. In addition, the results revealed that people felt relatively more connected and recalled more specific episodic events in the near future than distant future self condition, a finding that bolsters Wilson et al. ’s claim that the near future self is more important to the current self-regard than the distant future self [23]. Self-Appraisals in the Near and Distant Future In this stage, information is represented and analyzed more fully because more details including past or recent episodic experiences are referenced [27,29,32]. Also, the LPC effect has been linked to episodic memory retrieval and mental imagery [28,34–36]. For example, West, & Holcomb (2000) found an LPC effect was most evident between 550 and 800 ms in an imagery task relative to semantic decision and surface character- istics tasks [36]. Based on tenets of construal level theory that near future self-representations are more complex and comprised of both positive and negative attributes while distant future self- representations are more idealized and uniformly positive, it is possible that negative traits in the near future self condition evoked more specific episodic thoughts and imagery relative to negative traits evoked in the distant future self condition. This conjecture is consistent with behavioral results indicating participants recalled more specific events coming to mind and more perceived connectedness in the near future self condition compared to the context of a distant future. Author Contributions Furthermore, electrophysiological results revealing an LPC effect in the near future self condition but not in the distant future self condition suggest negative traits adjectives evoke more episodic thoughts in near future self-appraisals relative to distant future self- Conceived and designed the experiments: XH YL. Performed the experiments: YL XW. Analyzed the data: YL. Contributed reagents/ materials/analysis tools: YL. Wrote the paper: YL TJ XH. Conceived and designed the experiments: XH YL. Performed the experiments: YL XW. Analyzed the data: YL. Contributed reagents/ materials/analysis tools: YL. Wrote the paper: YL TJ XH. Discussion The current study examined both behavioral and neural responses related to processing of future self-appraisals as a function of temporal distance. Behavioral results showed people used fewer positive adjectives and more negative adjectives to describe their near future selves (i.e. one month from now) relative Table 2. Correlations between LPC amplitude and connectivity and event recall frequency in near and distant future self conditions. The LPC amplitude Connectivity Frequency Near Future Distant Future Near Future Distant Future Near Positive Future r .039 .026 .251 .07 p .878 .917 .315 .784 Near Negative Future r .063 2.027 .317 .215 p .804 .915 .2 .392 Distant Positive Future r .022 2.133 .095 2.124 p .932 .598 .709 .623 Distant Negative Future r .209 .086 2.042 2.094 p .404 .736 .867 .711 doi:10.1371/journal.pone.0084332.t002 PLOS ONE \| www.plosone.org 4 December 2013 \| Volume 8 \| Issue 12 \| e84332 etween LPC amplitude and connectivity and event recall frequency in near and distant future self Table 2. Correlations between LPC amplitude and connectivity and event recall frequency in near conditions. December 2013 \| Volume 8 \| Issue 12 \| e84332 Self-Appraisals in the Near and Distant Future 1. Szpunar KK (2010) Episodic Future Thought: An Emerging Concept. Perspect Psychol Sci 5: 142–162. 2. Schacter DL, Addis DR, Buckner RL (2008) Episodic simulation of future events: concepts, data, and applications. Ann N Y Acad Sci 1124: 39–60. Self-Appraisals in the Near and Distant Future Kanten A, Teigen K (2008) Better than average and better with time: Relative evaluations of self and others in the past, present, and future. Eur J Soc Psychol 353: 343–353. 32. Ito TA, Larsen JT, Smith NK, Cacioppo JT (1998) Negative information weighs more heavily on the brain: the negativity bias in evaluative categorizations. J Pers Soc Psychol 75: 887–900. 12. Macleod AK, Conway C (2005) Well-being and the anticipation of future positive experiences: The role of income, social networks, and planning ability. Cogn Emot 19: 357–374. y 33. Yuan JJ, Yang JM, Meng XX, Yu FQ, Li H (2008) The valence strength of negative stimuli modulates visual novelty processing: electrophysiological evidence from an event-related potential study. Neuroscience 157: 524–531. g 13. 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https://openalex.org/W2892782698	https://periodicos.furg.br/sinergia/article/download/7352/5208	Portuguese	null	O ESTADO E A GENERALIZAÇÃO DA FORMA EMPRESA: UMA ANÁLISE DA PERCEPÇÃO DE AGENTES PÚBLICOS ACERCA DO PAPEL DA SECULT EM PELOTAS/RS	Sinergia	2,018	cc-by	12,466	( ) ** Doutor em Administração pela Universidade Federal de Santa Catarina. Atualmente é Professor do Curso de Bacharelado em Administração, do Programa de Pós-Graduação em Sociologia e do Programa de Pós-Graduação em Desenvolvimento Territorial e Sistemas Agroindustriais da Universidade Federal de Pelotas. Também é líder do Núcleo de Estudos Marcelo Milano Falcão Vieira (NeMaVi). E GENERALIZATION OF THE ENTERPRISE FORM: AN ANALYSIS OF THE PERCEPTION OF PUBLIC AGENTS ABOUT THE ROLE OF SECULT IN PELOTAS / RS GENERALIZATION OF THE ENTERPRISE FORM: AN ANALYSIS OF THE PERCEPTION O PUBLIC AGENTS ABOUT THE ROLE OF SECULT IN PELOTAS / RS This study aimed to analyze how the state, represented here by the Department of Culture of the Municipality of Pelotas (Secult), contribute to the process of the enterprisation in the field of culture in the city of Pelotas. For this, we conducted a qualitative research, of descriptive-interpretative type, through a case study technique. By choosing a Foucaultian approach, in which the alignment to the neoliberal ideology contributes to the generalization of the enterprise form, we sought to describe how the changes occurred in guidance of the State on the field of culture. Thus, it was revealed the existence of neoliberal elements that characterize the action of the State and compose the process of enterprisation, mainly related to proclamations publication practices, decentralization and cultural training. This contributes to disseminate the idea of reproducing multiples individuals- enterprise composing the social tissue. Keywords: Enterprise. Culture. Neoliberalism. Enterprisation. Recebido em: 10-10-2017 Aceito em: 20-02-2018 RESUMO Este estudo buscou analisar como o Estado, representado aqui pela Secretaria de Cultura da Prefeitura Municipal de Pelotas (Secult), contribuicom o processo de empresarização no campo da cultura da cidade de Pelotas. Para isso, foi realizada uma pesquisa qualitativa, do tipo descritivo-interpretativa a partir da técnica de estudo de caso. Ao se optar por uma abordagem foucaultiana, na qual o alinhamento ao ideário neoliberal contribui para a generalização da forma empresa, buscou-se descrever como ocorreram as transformações na orientação do Estado frente ao campo da cultura. Diante disso, foi possível perceber a existência de elementos neoliberais que caracterizam a ação do Estado e compõe o processo de empresarização, principalmente relacionado à publicação de editais, de descentralização e de formação cultural, o que acaba por contribuir para disseminar a ideia de reproduzir múltiplos indivíduos-empresa, compondo o tecido social. Palavras Chave: Empresa. Cultura. Neoliberalismo. Empresarização. JANIELE CRISTINE PERES BORGES * MARCIO SILVA RODRIGUES ** JANIELE CRISTINE PERES BORGES * MARCIO SILVA RODRIGUES ** * Graduada em Administração e em Enfermagem pela Universidade Federal de Pelotas (UFPel), Mestra em Administração pela Universidade Federal de Rio Grande (PPGA/FURG) e Doutoranda em Administração na Universidade Federal do Rio Grande do Sul (PPGA/UFRGS). Recebido em: 10-10-2017 Aceito em: 20-02-2018 * Graduada em Administração e em Enfermagem pela Universidade Federal de Pelotas (UFPel), Mestra em Administração pela Universidade Federal de Rio Grande (PPGA/FURG) e Doutoranda em Administração na Universidade Federal do Rio Grande do Sul (PPGA/UFRGS). ** Doutor em Administração pela Universidade Federal de Santa Catarina. Atualmente é Professor do Curso de Bacharelado em Administração, do Programa de Pós-Graduação em Sociologia e do Programa de Pós-Graduação em Desenvolvimento Territorial e Sistemas Agroindustriais da Universidade Federal de Pelotas. Também é líder do Núcleo de Estudos Marcelo Milano Falcão Vieira (NeMaVi). Keywords: Enterprise. Culture. Neoliberalism. Enterprisation. 1 INTRODUÇÃO O foco desse estudo é no modelo neoliberal de atuação do Estado, que, ao contrário, utiliza as regras presentes na esfera econômica como uma espécie de tribunal econômico, em nome de uma lei de mercado que, dentre outras atuações, permitirá avaliar as ações do Estado segundo padrões de eficiência (FOUCAULT, 2008). Vale ressaltar que o surgimento do Estado neoliberal está relacionado, principalmente, à crítica a um Estado Keynesiano, no qual as políticas adotadas, o aparelho estatal de oferta de bens e serviços e o sistema de bem-estar social foram vistos como um pesado ônus ao Estado, que, por meio de sua autonomia política e institucional, realizava um investimento maciço em infraestrutura e em políticas sociais aos cidadãos. E é, a partir da crise desse modelo, que o Estado, ao olhar para a empresa, vê um modelo legitimado de eficiência, passando, então, a introduzir seus modos de gerenciamento e controle através do neoliberalismo (ARIENTI, 2003). E, no Brasil, o Estado neoliberal parece emergir no início dos anos 1990, marcado pela incorporação de um discurso há muito proferido pelos países centrais: esse esgotamento do Estado de bem-estar keynesiano e, consequentemente, o da necessidade de instituir o chamado Estado neoliberal schumpeteriano, na defesa de um Estado menos oneroso, mais flexível, orientado à empresa, à inovação tecnológica e ao trabalho, que se tornou um símbolo de modernidade e de progresso (ARIENTI, 2003). g q p g No neoliberalismo, o Estado, dotado de uma estrutura pública reduzida, adquire um papel de destaque como um agente regulamentador que contribui para a expansão da esfera privada. O neoliberalismo não procura a retirada do Estado, mas a transformação da ação pública, o que torna o Estado uma esfera regida pelas mesmas regras de concorrência e eficácia a que são submetidas às empresas privadas (LAVAL e DARDOT, 2016). Com a expansão da esfera privada e das leis da economia de mercado para decifrar e ordenar fenômenos antes não econômicos, a empresa surge como um modelo social universalmente generalizável, que serve tanto para organizações quanto para indivíduos (FOUCAULT, 2008). A ideia de empresarização, no Brasil, está presente em diferentes áreas, como, por exemplo, a educação (NEVES, 2002) e a geografia (VAINER, 2000). Entretanto, nos estudos organizacionais, ela ainda é pouco desenvolvida e está concentrada em um grupo relativamente pequeno de pesquisadores. 1 INTRODUÇÃO A definição de cultura é permeada por uma série de estudos que parecem gerar mais dúvidas e indagações do que certezas. O conceito adotado por Geertz (1989), a partir de uma perspectiva antropológica, está entre os mais aceitos e considera cultura como uma teia de significados que os indivíduos de uma sociedade constroem e reproduzem (GEERTZ, 1989). Além disso, é por intermédio da cultura que o homem cria, atribui sentido e transforma o seu mundo. A cultura contribui para representar e dar significado a determinado grupo de indivíduos, estabelecendo a forma como as coisas são feitas e aceitas e como os saberes são construídos (KNOPP et al, 2010). Por trás de toda essa representatividade e expressão, encontram-se não só indivíduos, mas organizações e instituições que atuam no campo e na transformação cultural. O estudo de Vieira e Simões (2010) mostra que o campo da cultura foi transformado, ao longo dos anos, pelas organizações culturais e, SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 81 principalmente, pelas ações e interesses da esfera pública, ainda que a atuação do Estado tenha ocorrido de forma descontinuada. Os períodos de ausência do Estado acabaram permitindo que outras organizações, como as do mercado, regulassem e interferissem no campo da cultura, transformando-o a partir da incorporação de elementos de fora do seu contexto. principalmente, pelas ações e interesses da esfera pública, ainda que a atuação do Estado tenha ocorrido de forma descontinuada. Os períodos de ausência do Estado acabaram permitindo que outras organizações, como as do mercado, regulassem e interferissem no campo da cultura, transformando-o a partir da incorporação de elementos de fora do seu contexto. p p Ainda que existam outros aspectos, supõe-se que o comportamento do Estado frente à cultura acaba por determinar a transformação no campo, seja pela maneira como ele age diretamente ou pela lacuna que deixa para que outros agentes preencham. O campo organizacional da cultura no Brasil se configurou de diferentes formas, inicialmente a cultura foi concebida e tratada como identidade, após como ideologia, mais adiante como estratégia e, atualmente, como mercado (VIEIRA e SIMÕES, 2010). g ( ) Uma aproximação entre Estado e mercado tende, tradicionalmente, a uma ideia de liberalismo econômico, que não se pretende abordar aqui, na qual a ação do Estado é limitada pelo mercado que age segundo suas próprias regras. 2 REFERENCIAL TEÓRICO Uma manifestação tipicamente moderna, o processo de empresarização está relacionado à compreensão da empresa como símbolo de eficiência, organização de referência e sistema normativo central das sociedades ocidentais e ocidentalizadas. Como um fenômeno social total (Solè, 2008), isto é, político, social, econômico, cultural e cognitivo; o processo de empresarização, caracterizado, aqui, como o avanço e predomínio da ideia de empresa sobre tudo e todos, contribui, no limite, para redefinir as relações sociais, as maneiras de viver e as subjetividades. É, nada mais e nada menos, a compreensão da empresa como um poder transversal que contribui para estabelecer a forma de nossa existência – o modo como nós agimos e nos relacionamos com os outros e com nós mesmos. Nesse processo, reside um poderoso discurso segundo o qual o “bom”, “aceitável” ou “positivo” é tudo aquilo que remete à ideia de uma organização empresarial. Essa compreensão da empresa como um modelo universalmente generalizável que permeia o tecido social parece estar sendo intensificado através do neoliberalismo. No Nascimento da Biopolítica, Foucault (2008) afirma que o neoliberalismo enquanto um conjunto de ideias e práticas assenta-se, dentre outros aspectos, em dois elementos centrais: (1) o uso da lente econômica para decifrar todo e qualquer fenômeno social; e (2) a compreensão da empresa como modelo e forma de organização central, amplamente difundida por todo tecido social, atingindo indivíduos e organizações. Neste trabalho, os elementos que balizaram o processo de análise estão diretamente alinhados à obra de Foucault (2008), na qual o autor traz diversos elementos característicos do neoliberalismo e representativos do processo de empresarização, são eles: autorresponsabilização do sujeito pela sua condição (como se fosse ele mesmo uma empresa); relações concorrenciais permeando o tecido social; Estado como agente mantenedor da ordem neoliberal; formação como instrumento para elevar o capital humano e centralidade no desenvolvimento da economia de mercado, tendo a inovação como principal instrumento. Esses elementos estão melhores descritos na sequência do texto. Na forma de atuação do Estado Neoliberal, mais do que o emprego de mecanismos produtivos, há uma preocupação com a forma com que são alocados esses recursos, há uma preocupação com a lógica que faz com que o indivíduo decida aplicar os recursos escassos em dado modo de produção e não em outro, para atingir um fim e não outro. 1 INTRODUÇÃO Impulsionado pelo neoliberalismo, parte-se da tese de que esse processo apresenta uma forma bastante sutil de reprodução e conta, assim como apontado em outros estudos, com o apoio das mais variadas instituições, dentre elas, o Estado, cuja lente econômica/empresarial é utilizada para traduzir, analisar e intervir em setores tradicionalmente não econômicos, como é o caso da cultura. Nesse sentido, o trabalho tem o objetivo de compreender de que forma o Estado, representado aqui pelos agentes públicos que atuam ou atuaram na Secretária Municipal de Cultura, contribui para generalizar a forma da empresa no campo da cultura da cidade de Pelotas. discussão sobre esse processo, que chama a atenção para a importância do Estado e para a maneira como agentes públicos, por estarem assim como todos nós, sujeitos a este poder, tendem a incorporar e reproduzir a ideia de empresa. Impulsionado pelo neoliberalismo, parte-se da tese de que esse processo apresenta uma forma bastante sutil de reprodução e conta, assim como apontado em outros estudos, com o apoio das mais variadas instituições, dentre elas, o Estado, cuja lente econômica/empresarial é utilizada para traduzir, analisar e intervir em setores tradicionalmente não econômicos, como é o caso da cultura. Nesse sentido, o trabalho tem o objetivo de compreender de que forma o Estado, representado aqui pelos agentes públicos que atuam ou atuaram na Secretária Municipal de Cultura, contribui para generalizar a forma da empresa no campo da cultura da cidade de Pelotas. p p O trabalho é divido em três seções além desta introdução e das considerações finais. A seção a seguir discorre sobre a abordagem teórico-analítica que orientou o desenvolvimento do trabalho. Após, são abordados os procedimentos metodológicos, para, finalmente, na terceira seção, desenvolver a análise dos resultados. Após esta terceira seção, são feitas algumas considerações finais à guisa de conclusão. O trabalho é divido em três seções além desta introdução e das considerações finais. A seção a seguir discorre sobre a abordagem teórico-analítica que orientou o desenvolvimento do trabalho. Após, são abordados os procedimentos metodológicos, para, finalmente, na terceira seção, desenvolver a análise dos resultados. Após esta terceira seção, são feitas algumas considerações finais à guisa de conclusão. 1 INTRODUÇÃO Os primeiros estudos sobre o processo de empresarização datam do início dos anos 2000 (COSTA, 2005; DURIEUX, 2005; SERRA, 2005; RODRIGUES, 2006; RODRIGUES e SILVA, 2006; HOFFMANN e DELLAGNELO, 2007).Partindo de um working paper disponibilizado por Andreu Solè (2004), que partia da noção da empresa como a organização central do mundo moderno, os trabalhos, naquela época,limitaram- se à descrição e à análise da intensidade com que as organizações esportivas, religiosas e culturais incorporavam um comportamento empresarial (RODRIGUES, SILVA e DELLAGNELO, 2014). p p p ( ) Após retornar àqueles trabalhos e perceber a abordagem (relacional) proposta por Solè (2004),apresentava diversos pontos descobertos (ver RODRIGUES, 2013), as discussões sobre o processo de empresarização adentraram em um segundo momento. A partir da proposta de Abraham (2006), buscou- se, além de ampliar as discussões do primeiro momento para o campo da educação (RODRIGUES, 2013) e da agricultura familiar (ARAÚJO; SILVA, 2016), evidenciar como a empresa, enquanto instituição, é construída e quais as implicações/desdobramentos do processo de empresarização. Para tanto, foram elaborados trabalhos orientados à análise da mídia no processo de construção e manutenção da centralidade da empresa no Brasil (RODRIGUES, 2013), à ênfase nos traços e relações modernas que fundamentam a ideia de empresa (RODRIGUES, 2013, ARAÚJO e SILVA, 2016; TOMETICH e SILVA, 2016; COSTA, 2017) e à discussão do empreendedorismo como um dos desdobramentos daquele processo (TAVARES e RODRIGUES, 2015; MARTINS, TAVARES e RODRIGUES, 2016; FRANZ e RODRIGUES, 2017). A busca por olhares que supram as lacunas apontadas pelos estudos anteriores é uma das motivações centrais deste texto. Busca-se, aqui, tanto contribuir com o avanço teórico da referida perspectiva, como abordar o papel das instituições no processo de construção e generalização da forma empresa em nosso mundo. Para tanto, adota-se uma perspectiva foucaultiana, até então inédita na 82 SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 discussão sobre esse processo, que chama a atenção para a importância do Estado e para a maneira como agentes públicos, por estarem assim como todos nós, sujeitos a este poder, tendem a incorporar e reproduzir a ideia de empresa. SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 2 REFERENCIAL TEÓRICO p p ç p O fato de a empresa representar um modelo social universalmente generalizado acaba por reconstituir uma série de valores morais e culturais, aproximando-os dos antiéticos, do mecanismo da concorrência, mas que garantem a sobrevivência desse modelo. A concorrência pode ser considerada o instrumento mais eficiente para elevar o desempenho da ação pública, “o homem neoliberal é o homem competitivo, inteiramente imerso na competição mundial (DARDOT e LAVAL, 2016, p. 322). Essas relações concorrenciais, inerentes ao ambiente empresarial e que atuam como um princípio no campo da economia de mercado, ao serem inseridas no meio social, tendem mais a atuar como um princípio dissolvente do que unificante. O Estado passa a não ser mais uma entidade que se situa fora da ordem do mercado, mas inteiramente integrado aos espaços mercantis e à interdependência dos agentes econômicos (DARDOT e LAVAL, 2016). Portanto, para que os mecanismos de concorrência possam agir no tecido social, torna-se necessária a atuação do Estado como mantenedor da ordem neoliberal, por meio de políticas que o mantenham acima das relações concorrenciais e que assegurem uma cooperação entre homens socialmente integrados. Com relação a isso, tem-se, na atualidade, a legislação como uma construção utilizada como instrumento para a manutenção desse Estado neoliberal (FOUCAULT, 2008). p ç ( ) O neoliberalismo, por intermédio de uma política orientada para a constituição do mercado, deixa subentendida uma série de objetivos, como evitar a centralidade, favorecer empresas médias, apoiar o artesanato e o pequeno comércio, multiplicar o acesso à propriedade, entre outros. A ideia é incluir os pequenos, fazê-los participar do mercado, torná-los empreendedores, ou seja, múltiplos indivíduos-empresa (FOUCAULT, 2008). Interessado em reproduzir na sociedade situações como o indivíduo empresário de si e a ideia de concorrência permeando as relações sociais, o Estado neoliberal encontra na formação desses indivíduos a peça-chave para manter essa ordem e garantir o desenvolvimento econômico. Conforme já foi mencionado acima, Foucault (2008) aponta para a ideia de que é o capital humano elevado que garantirá a elevação da renda. Isso, claro, relacionado ao fato de que são as novas combinações, defendidas por Schumpeter (1988), as promotoras do desenvolvimento econômico, ou como muito se tem ouvido falar, a inovação. Schumpeter (1988) e Foucault (2008), cada um à sua maneira, abordam a centralidade do empresário como promotor desse desenvolvimento econômico, pois a ele cabe a função de realizar a inovação. 2 REFERENCIAL TEÓRICO O Neoliberalismo ocupa-se da maneira como os indivíduos racionalizam o emprego dos recursos, visando atingir determinado fim, sendo esse fim o mantenedor da lógica econômica (FOUCAULT, 2008). Trazendo a definição de Robbins de que a economia é a ciência que estuda o comportamento humano como uma relação entre fins e meios. Foucault (2008) deixa a questão de qual cálculo faz com as pessoas, considerando os recursos raros, decidam atribuí-lo a um fim e não a outro, deixando a prerrogativa de que a economia do Estado neoliberal operacionaliza-se na racionalidade interna dos indivíduos. No modelo econômico neoliberal, existe uma estreita relação entre capital e competência, sendo capital o trabalhador capaz de gerar renda - relativamente maior, de acordo com a sua competência. Essa relação torna o trabalhador o responsável por melhorar a sua condição, ou seja, uma empresa para si mesmo. E é numa multiplicidade de empresas compondo o tecido social que o Estado neoliberal operacionaliza-se, com uma nova estrutura social, que faz do modelo econômico um modelo para as relações sociais e para a relação do indivíduo consigo mesmo e com a sociedade (FOUCAULT, 2008). O ponto central a ser destacado aqui diz respeito à articulação entre indivíduo, sociedade e Estado, operando de acordo com pressupostos ligados à ideia de homo economicus. O cálculo, a eficiência e a adequação estrita dos meios aos fins que se pretende alcançar passam a ser os determinantes da ação social, seja no âmbito do indivíduo, seja no âmbito público e na definição de políticas públicas. A ideia de 83 SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 “público”, assim, dá lugar a uma concepção atomizada de sociedade (SANTOS, 1987). “público”, assim, dá lugar a uma concepção atomizada de sociedade (SANTOS, 1987). p g pç ( ) Aqui é importante destacar a ascensão dos governos conservadores de Margareth Tatcher e Ronald Reagan na Inglaterra e Estados Unidos, respectivamente. Esses governos, na década de 1980, tiveram papel central em disseminar uma lógica centrada no indivíduo. A afirmação famosa de Tatcher dá ênfase a esta lógica atomizada: “essa coisa de sociedade não existe. O que há e sempre haverá são indivíduos”. Já Reagan afirmava, com frequência, que “os governos nunca são a solução para nada, mas sim parte dos problemas”. Ou seja, as soluções devem ser encontradas no âmbito privado, excluindo qualquer possibilidade de aprimoramento de instituições públicas. 2 REFERENCIAL TEÓRICO Para tanto, torna-se necessário que o Estado estabeleça com esse indivíduo-empresa uma relação educacional através de investimento em formação. A educação acaba tendo a finalidade de produzir agentes que se adaptam às necessidades da economia, e esses devem fazê-lo de forma autônoma (GAULEJAC, 2007). E, nesse contexto, incentivá-los a tomarem para si o papel de construtores da sua formação também é papel desse Estado neoliberal; afinal de contas, trata-se de uma multiplicidade de indivíduos atuando como se constituíssem, eles próprios, uma empresa. Diante disso, entende-se porque a centralidade da empresa, presente no íntimo dos membros de uma sociedade, é capaz de garantir a manutenção de uma ordem econômica neoliberal. E é, nesse sentido, que nos preocupa não somente o fato dos diversos tipos de organizações estarem assumindo a forma de empresa (RODRIGUES, 2006; 2013), mas dos indivíduos encurralados a assumir para si a responsabilidade de mudar a sua situação (FOUCAULT, 2008). 2 METODOLOGIA Para alcançar o objetivo proposto, optou-se por realizar uma pesquisa qualitativa, a qual busca, segundo Godoy (1995), identificar as causas das transformações e descrever suas implicações. Além disso, trata-se de uma pesquisa descritivo-interpretativa, a partir da técnica de estudo de caso, tendo como unidade de análise a Secretaria de Cultura da cidade de Pelotas. E, também, dado o objetivo da pesquisa, foi adotado um corte seccional com perspectiva longitudinal, no qual a coleta de dados é feita em um determinado momento, mas com resgate de dados e informações de períodos passados (VIEIRA e ZOUAIN, 2004). Originalmente, a intenção era analisar apenas a percepção dos agentes públicos que atuavam na Secult no momento da coleta de dados e, para isso, entrevistou-se o secretário de cultura de Pelotas (ENTREVISTADO 1) e dois assessores indicados por ele: o responsável pelos editais (ENTREVISTADO 2), SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 84 ator do mercado cultural; e o superintendente (ENTREVISTADO 3), ex-professor universitário na área de educação. O contato com essas percepções desencadeou um interesse em compreender a percepção dos secretários de cultura pregressos. Para tal, entrevistaram-se três ex-secretários de cultura (ENTREVISTADOS 4, 5 e 6), totalizando seis entrevistas. Vale ressaltar que, desde o período de criação da Secult, seis pessoas ocuparam o cargo de secretário de cultura, uma dessas ocupou o referido cargo em dois momentos distintos. Todavia, dois ex-secretários não estavam disponíveis no período de coleta dos dados. ator do mercado cultural; e o superintendente (ENTREVISTADO 3), ex-professor universitário na área de educação. O contato com essas percepções desencadeou um interesse em compreender a percepção dos secretários de cultura pregressos. Para tal, entrevistaram-se três ex-secretários de cultura (ENTREVISTADOS 4, 5 e 6), totalizando seis entrevistas. Vale ressaltar que, desde o período de criação da Secult, seis pessoas ocuparam o cargo de secretário de cultura, uma dessas ocupou o referido cargo em dois momentos distintos. Todavia, dois ex-secretários não estavam disponíveis no período de coleta dos dados. Ressalta-se que, no que diz respeito aos dados secundários, não foi possível obter acesso aos primeiros editais lançados e aos primeiros programas desenvolvidos. Nesse sentido, os dados secundários analisados são referentes à gestão passada, finalizada em 2016. O resgate histórico apresentado a seguir assentou-se, essencialmente, em entrevistas não estruturadas com os referidos entrevistados. 2 METODOLOGIA A opção por essa e não outra abordagem para a coleta de dados primários está intimamente relacionada à guinada teórica deste trabalho. Durante, aproximadamente, dez anos, um dos autores desse artigo tem se dedicado à análise do processo de empresarização de organizações tradicionalmente não empresariais, utilizando, basicamente, duas perspectivas teóricas complementares: uma que trata a empresa como uma a organização e outra que a trata como a instituição central no nosso mundo (ABRAHAM, 2004; SOLÉ, 2004). Em ambas, as categorias mencionadas pelos autores foram complementadas, revisadas e apropriadas à realidade das organizações brasileiras. Neste trabalho, ao se inserir uma abordagem foucaultiana para discutir esse processo, acreditou-se que seria relevante deixar que o campo, este e todos os outros analisados daqui para frente, nos demonstrasse como esse fenômeno, esse dispositivo de poder, opera. Uma vez que a intenção aqui não é apenas verificar a presença ou não de uma postura empresarial, mas, sim, desvendar como, lentamente, a ideia de empresa é disseminada e incorporada, como suas práticas transformam-se em valores e acabam por apontar aos sujeitos a única, senão, a melhor possibilidade. j p Para a análise dos dados, utilizou-se a técnica da análise de conteúdo, sendo que a mesma foi realizada em três fases: (1) a pré-análise, que se refere à organização do material obtido; (2) a descrição analítica, que consiste na codificação, classificação e categorização dos dados; e (3) o tratamento dos resultados, etapa em que ocorre a interpretação dos dados (BARDIN, 1988). Partindo dessa forma de análise, organizaram-se historicamente os dados coletados, ou seja, buscando uma linearidade dos fatos. E dentro de cada período histórico, buscou-se encontrar as categorias empresariais e neoliberais presentes. 3.1 Criação e organização da secretaria de cultura O projeto de criação da Secult foi concebido durante uma campanha de governo de um candidato da oposição, que buscou para representante do Estado na área da cultura um professor da Universidade Federal de Pelotas (UFPel), que no momento da posse acabara de retornar do doutoramento, e diz que aceitou o cargo por acreditar nas contribuições que poderia realizar no campo. Com a criação da Secult, pôde-se perceber uma movimentação para que as ações dessa Fundação fossem mapeadas e o cenário cultural minimamente organizado, em termos de ações, prédios históricos, teatros, museus etc. Percebe-se que, nesse período, o Estado tomou para si a responsabilidade de organizar e fortalecer o contexto cultural local, assumindo o papel de transformador do contexto, no qual suas ações vão ampliando o espectro da intervenção no cenário cultural pelotense. Tudo era recuperação porque nada havia, tudo foi criação e investimento. O museu da baronesa era uma degradação total. [...] Todo o acervo de arte dos nossos artistas, que haviam em diferentes momentos doado obras para a prefeitura, misturados com restos de Kombi, pneus, pedações de madeira, tijolo, debaixo de um telhado todo esburacado [...] transformar o entulho, dar dignidade ao entulho (ENTREVISTADO 4). A centralidade do Estado é evidente nesse contexto de estruturação, com projetos criados, controlados e mantidos pelo Estado, sem interferências notórias da esfera econômica. Havia também uma preocupação com a criação e manutenção de projetos extensivos que se repetissem ano após ano, o que representa interesse na manutenção das atividades, sem evidências de direcionamento para a inovação. Fato evidenciado na fala abaixo: O que nós tratamos de fazer, a equipe que foi pra secretaria, foi justamente construir uma série de projetos separados por áreas de atuação, conforme as linguagens da cultura e da arte, que pudessem ser projetos extensivos, que pudessem acontecer todos os anos [...] então se montava projetos que ora eram estimulados por demandas da comunidade, ora por essa inteligência toda que a gente reuniu dentro da secretaria (ENTREVISTADO 4). No que diz respeito aos projetos com financiamento federal, nota-se uma centralização das ações na esfera pública e aumento do aparelho estatal, o qual incorporava para si a execução dos projetos existentes. 3 DESENVOLVIMENTO Como forma de reconstruir as transformações que ocorreram na maneira de atuação do Estado frente ao cenário cultural pelotense, o contexto de análise foi dividido em três períodos. O primeiro deles, o período de criação e organização da secretaria de cultura, que ocorreu de 2001 a 2004, para o qual foi realizada entrevista com o secretário de cultura da época. O segundo, caracterizado por um período de modernização do campo, para o qual foram entrevistados dois ex-secretários: um deles atuou em dois momentos separados, de 2005 a 2007 e de 2013 a 2014, e o outro somente no ano de 2012. E o terceiro, em que ocorreu a intensificação das ações, com agentes do mercado representando o Estado e fomentando ações, e para o qual, além do secretário de cultura, foram entrevistados dois funcionários indicados pelo secretário. p Um dado relevante é que, anterior à criação da Secult, existia a Fundação Municipal de Cultura, Lazer e Turismo de Pelotas, responsável até então pelas ações que o Estado realizava no cenário cultural. Diferentemente do que ocorreu no governo federal, no qual, antes da criação do Ministério da Cultura, as ações culturais eram tratadas em conjunto com a educação. Em Pelotas, essa fundação, como é possível perceber no próprio nome, tinha suas ações culturais ligadas ao lazer e turismo. Trata-se de uma forma de conceber a cultura que pode contribuir para delimitar, de certa forma, esse cenário cultural. Como diz Dória (2001), é o Estado que acaba por definir e fixar, em dado momento histórico, o que aquela sociedade entenderá por cultura. p Ao definir que a cultura representa um conjunto de saberes e fazeres de uma sociedade (VIEIRA, SILVA e RODRIGUES, 2010) e é elemento fundamental e insubstituível na construção da própria identidade nacional (BRASIL, 2015), seria esperado que as ações culturais estivessem ligadas à educação, pois é essa que nos transmite a ideia de formação e transformação. Situação essa que foi levantada no relato abaixo: O que significa o setor de cultura da cidade estar vinculado a uma secretaria de educação ou estar numa fundação autônoma vinculada a desporto, lazer e turismo? São duas formas de propor projetos e gerenciar tudo isso muito díspares e determinadas por um mesmo posicionamento político (ENTREVISTADO 4). SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 85 3.1 Criação e organização da secretaria de cultura Essa situação é claramente evidenciada na forma como a secretaria se estruturou para dar início às atividades do Programa Monumenta, para o qual recebeu um convite do Ministério da Cultura e do Instituto do Patrimônio Histórico e Artístico Nacional (IPHAN), uma das vinte e seis cidades brasileiras selecionadas. Para o desenvolvimento desse projeto, foi selecionada uma equipe de arquitetos que trabalhariam dentro da secretaria. Isso ocorreu de forma diferenciada ao que foi visto nas outras cidades, as quais elegeram escritórios terceirizados que lhes apresentaram projetos para o restauro dos prédios. q g q p p j p p Esse fato relaciona-se com a realização de concurso para a contratação dos arquitetos, com a intenção de que esses permanecessem na secretaria, fortalecendo o papel do Estado com uma equipe permanente e própria na elaboração desses projetos, não entregando ao mercado esse papel. Além disso, em uma organização pública, cujo restante dos funcionários é substituído a cada mandato político, a permanência dos arquitetos representa a constituição de um aparato do Estado. Essa situação contraria a lógica neoliberal, que prega a diminuição do tamanho do Estado com consequente transformação das suas funções (ARIENTI, 2003; DARDOT e LAVAL, 2016). ç ( , ; , ) No que confere ao papel da cultura, pode-se observar que o Estado, durante esse período, adotou um papel de agente transformador do indivíduo, fato evidenciado na convergência das falas do secretário quanto aos objetivos da Secult. O papel da cultura e sua contribuição para o desenvolvimento estão, respectivamente, listados abaixo: Tu queres que o cidadão comum tenha acesso ao que há de melhor no mundo, era isso que a gente queria. Eu queria que eles tivessem, que todo mundo, que nós tivéssemos acesso (ENTREVISTADO 4). q q q q g q queria que eles tivessem, que todo mundo, que nós tivéssemos acesso (ENTREVISTADO 4). [cultura] como uma possibilidade de revolução que nós temos nesse mundo desgraçadamente capitalista, [...] nos transforma em outras pessoas, essa ideia do cultivo, uma perspectiva frente à vida que nos coloca como construtores da vida e não como herdeiros subalternos de um conjunto de valores que nos acachapa toda hora (ENTREVISTADO 4). 3.2 O impulso modernizante Com o início de um novo mandato político e a nomeação de um produtor cultural para o cargo principal da Secult, verifica-se uma mudança na forma de como o Estado compreende a cena cultural pelotense. Como ilustra a fala abaixo: Até então nós tínhamos pleitos que já tinham sido feitos em encontros de culturas que nós fazíamos, nós que eu digo os produtores culturais, agentes de cultura. No governo [anterior] nós pleiteamos muito uma conferência municipal de cultura, o que nunca aconteceu, um Conselho Municipal de Cultura. Então nós tínhamos pleitos importantes e quando eu me vi naquela situação de secretária eu achei que eu tinha obrigação de imediatamente implementar essas [...] vamos dizer assim, abrir o espaço para a sociedade civil (ENTREVISTADO 5). Paralelamente a essa mudança que ocorria no cenário municipal, em nível nacional, já‟ havia a previsão de um Plano Nacional de Cultura (PNC), que só foi promulgado pela Lei 12.343 em 2010. Esse plano estabelece uma série de exigências para os municípios, com vistas a viabilizar o recebimento de verbas do governo estadual e federal. Essa movimentação em nível nacional corroborou para a reestruturação do cenário cultural pelotense, o qual passou a ser organizado para convergir com o PNC. Em 2005, o então governo da cidade de Pelotas declarou seu interesse em participar do PNC, durante um evento que ocorreu em Porto Alegre, na presença do então Ministro da Cultura Gilberto Gil. Essa situação deixou implícita a necessidade de implementação das conferências municipais de cultura e o estabelecimento de programas de incentivo à cultura. As ações eram direcionadas diretamente ao Plano Nacional de Cultura e, consequentemente, ao Sistema Nacional de Cultura. Ainda assim, para além da necessidade de se adequar às exigências do governo federal, o entrevistado 5 declarou que algumas dessas ações, como os editais de incentivo à cultura, já estavam presentes nos seus planos, de forma independente do que ocorria no cenário nacional: “isso tava, já, vamos dizer assim, na minha proposta e na proposta do prefeito, que era estabelecer essa relação de proximidade com a sociedade civil e dando poder à sociedade civil de decidir onde colocar os recursos públicos, isso sempre foi a bandeira do prefeito” (ENTREVISTADO 5). 3.1 Criação e organização da secretaria de cultura Eu acho que o desenvolvimento é decorrência, porque esse sujeito mais cultivado, não vou dizer mais culto porque se confunde com o cara que tem acúmulo de informações e de dados de cultura, esse sujeito mais q q q q ( ) [cultura] como uma possibilidade de revolução que nós temos nesse mundo desgraçadamente capitalista, [...] nos transforma em outras pessoas, essa ideia do cultivo, uma perspectiva frente à vida que nos coloca como construtores da vida e não como herdeiros subalternos de um conjunto de valores que nos acachapa toda hora (ENTREVISTADO 4). E h d l i é d ê i j i i l i d ã di i l ( ) Eu acho que o desenvolvimento é decorrência, porque esse sujeito mais cultivado, não vou dizer mais culto porque se confunde com o cara que tem acúmulo de informações e de dados de cultura, esse sujeito mais cultivado, ele vai ser um cidadão muito mais autônomo, mais crítico. Pra mim é uma decorrência obvia, e que é o que nos distingue daquilo que ainda se chama de centro, centro e periferia (ENTREVISTADO 4). De forma geral, os primeiros anos da Secult são marcados pelo esforço e intervenção do Estado no processo de organização e estruturação de alguns elementos que compõem a cena cultural pelotense, SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 86 como museus, teatros e prédios históricos. Segundo o entrevistado, tudo isso foi realizado com o intuito de contribuir com a transformação social através da construção de sujeitos críticos e mais conscientes da sua condição. É interessante notar, também, que, nesse período, a ideia de economia não se apresenta como uma máxima, inclusive não foi mencionado nenhum contato com atores do mercado cultural. Aliás, ela sequer é mencionada pelo entrevistado. como museus, teatros e prédios históricos. Segundo o entrevistado, tudo isso foi realizado com o intuito de contribuir com a transformação social através da construção de sujeitos críticos e mais conscientes da sua condição. É interessante notar, também, que, nesse período, a ideia de economia não se apresenta como uma máxima, inclusive não foi mencionado nenhum contato com atores do mercado cultural. Aliás, ela sequer é mencionada pelo entrevistado. 3.2 O impulso modernizante Escrevam um projeto, deixem o Conselho Municipal de Cultura dizer: 'do montante que a secretaria tem disponível, nós julgamos mais importante investir tanto aqui, tanto ali, tanto ali (ENTREVISTADO 5). A secretaria tá acabando com toda e qualquer participação do executivo [Estado], de forma que seja a mais democrática possível (ENTREVISTADO 5). Com relação à criação do Dia do Patrimônio, outra ação instituída, concebida pelo entrevistado 5 como uma atividade, cuja secretaria não executa as ações, somente estimula para que grupos e indivíduos tragam as atividades, sendo assim, a comunidade é que desenvolve as atividades e o Estado as regula, fornece equipamentos e organiza horários e divulgação. Outro ponto levantado pelo entrevistado é a necessidade de retornar ao mercado através da sua empresa. E foi por esse motivo, depois de um ano e meio atuando pela segunda vez como secretário, que o entrevistado solicitou exoneração. E na fala abaixo, fica evidente a relação estabelecida entre a ex- secretária, sua empresa e o mercado em que se insere: “eu só não consegui permanecer porque na minha empresa, se eu paro de trabalhar por quatro anos, é muito difícil eu conseguir retomar depois, se eu me afasto do mercado de trabalho por tanto tempo” (ENTREVISTADO 5). Ao sair da secretaria, as ações que estavam em andamento seguiram-se, sem qualquer prejuízo, pois um dos seus assessores assumiu o cargo de secretário, mantendo juntamente com ele, todo o pessoal que lá já estava. Para fazer referência à manutenção dos assessores, a entrevistada 5 usa o termo “técnicos em cultura”, referindo-se ao pessoal que atualmente trabalha na secretaria: “disseram que todos seriam mantidos, que aquela secretaria continuaria uma secretaria sem o cunho partidário que muitas têm nos governos, é natural que tenha. A Secult é formada por técnicos em cultura, não tem esse perfil de cargos que sejam indicados por políticos coisa desse tipo” (ENTREVISTADO 5). q j p p p ( ) É possível perceber uma alteração na relação entre Estado e cenário cultural pelotense. O financiamento das ações culturais, que antes ocorria mediante relação direta entre a Secult e os artistas, que lentamente os coloca, em competição passou a ser estabelecido por editais. Uma competição na qual os critérios de avaliação das propostas estão, de certa forma, orientados por elementos econômicos, estabelecendo para o Estado o papel de modernizador do campo. 3.2 O impulso modernizante ( ) As ações desenvolvidas, nesse período, foram a ativação do Conselho Municipal de Cultura (Concult), inclusive com a reestruturação do mesmo; a redação da legislação do Procultura municipal, a qual não foi implementada nessa gestão; e as conferências municipais de cultura. O programa de incentivo à cultura municipal (Procultura) ficou nesse período limitado apenas à redação, pois, mesmo com todo o esforço que o entrevistado 5 diz ter feito, não obteve êxito para a sua aprovação. E este foi um dos motivos que a levou a exonerar-se do cargo em 2007: uma série de desentendimentos com o novo prefeito, que assumira o cargo após o afastamento do seu antecessor. Não obstante, o Procultura foi aprovado no ano de 2009, quando o entrevistado já não atuava mais como secretário de cultura, mas como presidente do Concult. Nessa nova posição, realizou um esforço para que o prefeito promulgasse a lei de incentivo à cultura, a qual garantiria o destino de uma determinada verba à publicação para os editais de incentivo. Situação essa que pode ser evidenciada na fala do entrevistado 5: “eu fui Presidente do Conselho Municipal de Cultura, e aí o Conselho Municipal de Cultura cobrava do poder executivo a implementação do Procultura” (ENTREVISTADO 5). ( ) Após a saída do entrevistado 5, ocuparam o cargo dois secretários de cultura, aos quais não se obteve acesso, no período entre 2007 e 2012. Entre os meses de abril e dezembro de 2012, atuou como secretário de cultura o entrevistado 6, o qual, vale ressaltar, trabalhava na secretaria desde 2005. Essa última situação nos auxiliou a compreender o período que se teve acesso aos secretários de cultura. Trata- se de um período cuja esfera econômica manteve sua centralidade, inclusive pela fala do entrevistado 6, quando se referiu à relação entre cultura e desenvolvimento: Eu entendo que a cultura é uma das áreas que mais movimenta a questão econômica, porque, vamos pensar, só o carnaval, vamos pegar o carnaval. O que que tu tem? Tu tem desde o alfinete, a agulha, as lantejoulas, então tu tá movimentando a costureira, o serralheiro, o comerciante que vende os aviamentos, o pessoal que coloca a música, que bora o som, que bota a luz, tu movimenta todos os setores da economia em pequena escala e não em grande escala. 87 SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 3.2 O impulso modernizante 2017 87 Em um segundo momento em que o entrevistado 5 esteve na função de secretário, cargo para o qual retornou após a troca de mandato político na prefeitura, houve uma preocupação com a ampliação dos programas de incentivo à cultura, mediante publicação de editais. A ideia e até mesmo a redação de projetos implementados apenas na gestão seguinte foram realizadas nesse período, como é o caso do Prêmio Movimento e do edital de auxílio para eventos. O Prêmio Movimento foi idealizado, segundo o entrevistado 5, a fim de contemplar as pessoas que não tinham condições de elaborar um projeto “bem instruído, bem escrito e bem orçado”, como exige o Fundo Municipal de Cultura. Esse edital foi escrito com a intenção de chegar a pessoas da comunidade de Pelotas que, conforme relatou o entrevistado 5, "exercem liderança nas suas comunidades", mas que muitas vezes não sabem escrever. Trata-se de um edital que permite ao candidato se inscrever de diversas formas, inclusive de forma verbal. Há o reconhecimento, por parte do entrevistado 5, de que os editais que deveriam democratizar, acabam falhando, pois fazem aos candidatos uma série de exigências, impedindo o acesso de todos os que poderiam concorrer e receber apoio oficial. Vale ressaltar que, nesse período, o que ocorreu foi apenas a idealização e a redação desse edital, o qual foi implementado somente na gestão seguinte. g O edital referente a eventos também foi idealizado, embora tenha sido lançado em 2015. Essa idealização reforça o intuito de fornecer apoio financeiro mediante editais de concorrência, ainda que, como afirma o entrevistado 5, diga respeito a eventos importantes para a sociedade. Fato esse que pode ser evidenciado nas falas abaixo: [situação] que também foi gestada naquele tempo, que era não se dar mais apoios a eventos, mesmo que eventos reconhecidos pela comunidade como têm festas e várias coisas que acontecem anualmente [...] eventos cívicos. Não se oferecer mais apoio sem que os projetos passem por uma seleção também, mesmo que sejam eventos reconhecidos. É chato, aquela coisa da pessoa ir, sentar e dizer assim: „ah, eu tô fazendo o 20 de setembro, nós vamos fazer isso e aquilo e precisamos de tanto‟. „Ah, nós vamos fazer a via sacra, que é uma coisa super importante também e precisamos disso, disso, disso e daquilo‟. 3.2 O impulso modernizante E assim, o papel assumido pela Secult aproximou o Estado da lógica econômica, conformando uma forma de atuação antes não presente ou não importante, mas que agora confere ao Estado o papel de mantenedor de uma lógica neoliberal. Segundo Foucault (2008), no neoliberalismo, o Estado ocupa um papel central, inserindo nos mais diversos contextos sociais, tal como o cultural, elementos da economia de mercado. A centralidade das ações parece ser encontrada na implementação dos editais, que garantem a manutenção de um mercado e a incorporação da forma de atuação da empresa por parte dos indivíduos. No tocante a essa disseminação dos editais, há o reconhecimento de que diversos atores da cena cultural são desprivilegiados, como aqueles que não sabem escrever e são considerados merecedores de apoio. Na SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 88 tentativa de sanar essa falha, a Secult cria então o Prêmio Movimento, aproximando do estado e dessa esfera quem antes estava distante. Alguns pontos importantes reforçam a presença da forma de ser da empresa: a formalização dos editais, que impõe aos concorrentes uma atuação delimitada, como delimitam as empresas a manifestação individual dos seus trabalhadores por meio das exaustivas normas de comportamento; a tecnificação esperada dos membros que compõe a Secult e a ideia de concorrência na busca de melhorias. tentativa de sanar essa falha, a Secult cria então o Prêmio Movimento, aproximando do estado e dessa esfera quem antes estava distante. Alguns pontos importantes reforçam a presença da forma de ser da empresa: a formalização dos editais, que impõe aos concorrentes uma atuação delimitada, como delimitam as empresas a manifestação individual dos seus trabalhadores por meio das exaustivas normas de comportamento; a tecnificação esperada dos membros que compõe a Secult e a ideia de concorrência na busca de melhorias. Outro elemento importante é o fato de que o entrevistado 5, por atuar no mercado cultural, já buscava recursos para os projetos particulares de sua empresa em editais estaduais e nacionais. Então, em função dessa atuação e conhecimento do contexto de mercado vivenciado, o entrevistado 5 apresenta uma capacidade de antecipação das ações e de previsão do que acontece nas demais esferas governamentais. Essa situação possibilitou o desenvolvimento de um programa de incentivo à cultura, através da publicação de editais, antes mesmo que a prefeitura da cidade de Pelotas recebesse uma orientação nacional ou estadual. 3.3 Intensificação das ações, participação e fomento A transição para esse novo período é representada pela mudança na figura do secretário de cultura. A transição do período anterior para esse parece ter ocorrido de forma tranquila, visto que o restante do pessoal foi mantido. Claro que, tudo isso, é para dar seguimento ao modelo de gestão estabelecido. p q p g g Esse modelo parece estar centrado, principalmente, na publicação de editais, com a publicação de um número cada vez maior para diferentes propósitos, como o programa municipal de incentivo à cultura (Procultura), os de seleção de apresentações (Sete ao Entardecer e Palco no Parque), os de apoio a eventos e o do prêmio de reconhecimento à cultura popular (Prêmio Movimento). O modo de atuação da Secult, baseado na publicação de editais, é definido pelo atual secretário como uma forma democrática de fracionar os recursos da cultura. Fato esse que pode ser percebido nas falas abaixo: A política cultural da nossa gestão é de procurar cada vez mais democratizar o acesso tanto à cultura quanto aos recursos que a secretaria dispõe pra fomentar as ações culturais da cidade [...] estamos já praticamente aumentando o lançamento dos editais (ENTREVISTADO 1). A forma que nos encontramos foi exatamente a de publicar editais, porque isso faz com que a pessoa se prepare pra isso. [...] fala e não tem um projeto, não tem algo escrito, não tem um histórico, as pessoas precisam se organizar, hoje não é mais possível chegar e „minha arte fala por mim mesmo‟, até fala dependendo da situação. Mas, em qualquer lugar que ele vá buscar um financiamento, ele tem que ter o seu projeto, ele tem que saber porque faz e como faz. Então eu vejo que a forma de incentivar é cada vez mais publicar diferentes possibilidades da pessoa concorrer ao financiamento, ao apoio e a ajuda pra desenvolver a sua arte (ENTREVISTADO 3). ( ) A gente está fomentando, incentivando pra que diferentes áreas, pra que diferentes segmentos se organizem pra oferecer ações. E que forma nós achamos para incentivar? Com a publicação de editais (ENTREVISTADO 3). Em todos os editais, apenas um determinado número de projetos será contemplado, gerando uma espécie de relação concorrencial entre os candidatos, semelhante à forma com que empresas se relacionam em determinado mercado. Na busca pela aprovação dos projetos, é necessário que os candidatos estejam atentos ao edital e aos critérios que serão pontuados na avaliação. 3.2 O impulso modernizante A competição, colocada como estratégia para democratizar o acesso aos recursos culturais, já ocorria em nível nacional e estadual, ou seja, o Estado incentivando a concorrência entre os atores, como se esses fossem empresas competindo em busca da sua sobrevivência em um determinado mercado. 3.3 Intensificação das ações, participação e fomento Diante disso, o Estado desperta a ideia de competição entre os atores da cena cultural pelotense e deixa de privilegiar determinados projetos para atender a essa política, como se verifica nas falas abaixo: Quem entra pra participar sabe onde é que ele precisa se mostrar mais (ENTREVISTADO 2). Quem entra pra participar sabe onde é que ele precisa se mostrar mais (ENTREVISTADO 2). Os editais fazem com que a pessoa se prepare para concorrer (ENTREVISTADO 3). Não é uma tarefa fácil, porque às vezes a gente tem que deixar de aprovar projetos bons, reconhecidamente bons, mas em função do limite de verba não se pode atender a todos, então tu tem que chegar a algum momento que tu tens que cortar (ENTREVISTADO 3). Os editais fazem com que a pessoa se prepare para concorrer (ENTREVISTADO 3). Não é uma tarefa fácil, porque às vezes a gente tem que deixar de aprovar projetos bons, reconhecidamente bons, mas em função do limite de verba não se pode atender a todos, então tu tem que chegar a algum momento que tu tens que cortar (ENTREVISTADO 3). O que chama a atenção no edital de incentivo à cultura são os critérios utilizados para avaliar os concorrentes: o ineditismo, o mérito artístico; a qualificação do proponente; os desdobramentos e impactos capazes de gerar resultados no campo cultural, social e econômico; entre outros. Critérios esses muito próximos dos encontrados nos editais para seleção de apresentações culturais, como, por exemplo: originalidade, a qual subentende trabalhos inovadores; e currículo, que diz respeito à qualificação dos profissionais envolvidos. O edital para eventos também possui critérios nesse sentido, como autossustentabilidade, critério que avalia o potencial de autossustentabilidade nas edições vindouras e, 89 SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 também, o critério de democratização do acesso que possui como subcategoria a realização de atividades fora do centro, descentralizadas (PELOTAS, 2015). também, o critério de democratização do acesso que possui como subcategoria a realização de atividades fora do centro, descentralizadas (PELOTAS, 2015). Apesar da continuidade, um novo elemento parece ganhar espaço na narrativa dessa gestão, a descentralização, vista como uma meta dessa gestão, pois segundo o secretário “descentralizar é uma necessidade” (SANGUINÉ, 2014). Fato que pode ser evidenciado na fala abaixo: A nossa urgência agora, na verdade, é chegar nos bairros (ENTREVISTADO 1). A nossa urgência agora, na verdade, é chegar nos bairros (ENTREVISTADO 1). O Piquenique Cultural, por exemplo, [...] uma ação que tem muito mérito como uma ação independente (ENTEVISTADO 1). Então a gente gosta, na verdade, é de estimular que a comunidade carnavalesca procure recursos [...] com o passar dos anos talvez o carnaval fique independente do poder público (ENTREVISTADO 1) Incentivando ações independentes e descentralizadas, como uma multiplicidade de empresas agindo no ambiente, a Secult parece transformar-se, pois, por intermédio de seus representantes, atua como “fomentador”, “inovador” e através de “conexões” entre ofertas e demandas de ações culturais. Situação essa que evidencia o Estado como agente regulador no contexto, por meio de agentes do mercado e para o mercado. Além disso, parece introduzir, no contexto cultural, termos como “trabalhador cultural”, que não era característico desse contexto, como se pode apreender abaixo: Porque, pelo menos como a gente vem da iniciativa privada, a gente tá acostumado a fazer projeto com pouquíssimo recurso, a gente é mais um trabalhador da cultura que nesse momento está aqui entendeu?! Então a gente acaba tendo um pouco essa visão e aí de funciona como facilitador mesmo, é isso [...] Então, trabalhar em rede com o trabalhador da cultura mesmo, acho que é isso que tem nos feito transitar bem nos seguimentos. Como é que a gente mapeia isso? Olha a gente mapeia ouvindo, recebendo demanda, atento ao que tá acontecendo (ENTREVISTADO 2). As secretarias de cultura, elas ficaram estigmatizadas durante muito tempo, como apoiadoras financeiras de eventos. [...] Então, nós não somos isso, não somos uma secretaria que paga contas, nós nos enxergamos como fomentadores de ações culturais, em toda a sua amplitude (ENTREVISTADO 1). Além dessa parte de editais a gente faz [...] a gente trabalha bastante estabelecendo conexões [...] conexão na verdade, como um grande modo de operação nosso (ENTREVISTADO 1). Porque, pelo menos como a gente vem da iniciativa privada, a gente tá acostumado a fazer projeto com pouquíssimo recurso, a gente é mais um trabalhador da cultura que nesse momento está aqui entendeu?! Então a gente acaba tendo um pouco essa visão e aí de funciona como facilitador mesmo, é isso [...] Então, trabalhar em rede com o trabalhador da cultura mesmo, acho que é isso que tem nos feito transitar bem nos seguimentos. Como é que a gente mapeia isso? Olha a gente mapeia ouvindo, recebendo demanda, atento ao que tá acontecendo (ENTREVISTADO 2). A nossa urgência agora, na verdade, é chegar nos bairros (ENTREVISTADO 1). A gente acredita que no ano passado a gente já conseguiu fazer umas ações descentralizadoras da cultura e esse ano a gente quer ainda mais (ENTREVISTADO 1). Outra forma encontrada de descentralizar foi o Prêmio Movimento, o qual previa, por meio da publicação de edital, a inscrição de agentes com iniciativas culturais fora do centro da cidade, ou seja, era proibida a inscrição de ações que ocorressem no centro. Um programa que, embora tenha sido idealizado na gestão anterior, parece ter adquirido nesta gestão o caráter local, e que, de certa forma, pode representar uma forma de mapeamento e desenvolvimento das ações locais. Aliando a ideia de descentralização à de formação, a Secult está estruturando um Programa de Formação Cultural, o qual pretende, a partir de demandas específicas da comunidade, distribuir agentes culturais nos bairros. Além disso, com uma ideia de complementariedade, esses cursos foram idealizados para serem oferecidos nas áreas que não são atendidas pelas universidades da cidade. Além desse programa de formação cultural, a Secult realiza um curso de prestação de contas para os habilitados no Procultura e outro curso para os inabilitados na primeira fase do Procultura, esclarecendo porque não continuaram a concorrer e como podem melhorar os seus projetos para as próximas inscrições. Além disso, há uma proposta de que os editais municipais preparam os atores para editais de outras esferas governamentais, como os estaduais e federais. Situação evidenciada nas falas abaixo: Os nossos editais dão uma preparação pra que os agentes culturais possam se inscrever em editais estaduais e federais (ENTREVISTADO 1). A secretaria de cultura valoriza a arte e a cultura daquilo que é bom e tenta fomentar pra que as pessoas desenvolvam o seu potencial (ENTREVISTADO 3). cretaria de cultura valoriza a arte e a cultura daquilo que é bom e tenta fomentar pra que as pessoa nvolvam o seu potencial (ENTREVISTADO 3). Essas melhorias e capacitações parecem repercutir em profissionalização e independência dos artistas. O desejo do Estado por essa independência surge na redação do edital para eventos que pontua a capacidade de autossustentabilidade para os próximos eventos, como demonstrado acima. Essa situação está diretamente relacionada à defesa do Estado em prol das ações culturais independentes, situação evidenciada nas falas que seguem: Se a gente prestar atenção nos programas independentes, eles são todos um sucesso: o Sofá na Piquenique Cultural, Samba no Mercado (ENTREVISTADO 1). 3.3 Intensificação das ações, participação e fomento A gente acredita que no ano passado a gente já conseguiu fazer umas ações descentralizadoras da cultura e esse ano a gente quer ainda mais (ENTREVISTADO 1). A nossa urgência agora, na verdade, é chegar nos bairros (ENTREVISTADO 1). A nossa urgência agora, na verdade, é chegar nos bairros (ENTREVISTADO 1). ( ) As secretarias de cultura, elas ficaram estigmatizadas durante muito tempo, como apoiadoras financeiras de eventos. [...] Então, nós não somos isso, não somos uma secretaria que paga contas, nós nos enxergamos como fomentadores de ações culturais, em toda a sua amplitude (ENTREVISTADO 1). Além dessa parte de editais a gente faz [...] a gente trabalha bastante estabelecendo conexões [...] con verdade, como um grande modo de operação nosso (ENTREVISTADO 1). SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 90 Surge, também, a preocupação com a modernização, a inovação da sua própria forma de agir, pois, segundo o entrevistado 1, a secretaria está “sempre revendo o modo de operação”. Assim como nos editais, o ineditismo e a originalidade são critérios pontuados, na forma de agir da Secult esses também são critérios importantes. E revendo o seu modo de operação com base em modelo fora do cenário da cultura, nesse caso o empresarial, situação percebida na forma como atua no contexto cultural e como define os critérios de avaliação dos editais. ç Outro critério, ao qual o Estado parece dar atenção, são os desdobramentos gerados a partir das ações culturais. A atuação no campo em si já não parece ser suficiente para o Estado, é preciso gerar resultados no campo cultural, social e econômico, como demonstra um dos critérios do Procultura. No entanto, parece que o viés econômico tem assumido um papel importante nessas ações, parece ser para onde caminham as demais ações, como defendido pelo entrevistado 3: Cultura como expressão simbólica, como expressão cidadã e dimensão econômica. Então é a cultura se fundamentando, expressão simbólica porque ela tá no subjetivo de todas as pessoas, cidadã porque é isso né reconhecimento daquilo que a pessoa faz, realiza e é capaz, e a dimensão Econômica [...] no que ela está baseado e o que ela pode suscitar. [...] Aí entra o lado econômico, a cultura ela é capaz de gerar renda, ela é capaz de permitir que as pessoas se mantenham por elas mesmas, então é a questão da profissionalização dos artistas (ENTREVISTADO 3). Durante esse período, estava sendo construído o Plano Municipal de Cultura, o qual estava alinhado ao Plano Nacional de Cultura, e, como exigência desse, trata-se de um projeto com duração de dez anos. “São dez anos porque tá ligado ao Plano Nacional de Cultura, é uma exigência do Plano Nacional de Cultura” (ENTREVISTA 3). A nossa urgência agora, na verdade, é chegar nos bairros (ENTREVISTADO 1). É importante atentar para o fato de que muitas das narrativas encontradas estão diretamente relacionadas ao fato da Secretaria Municipal de Cultura estar buscando a construção do Sistema Municipal de Cultura, o que exige a elaboração do Plano Municipal de Cultura e permitirá a inserção nos sistemas estadual e nacional de cultura. Como argumenta o Entrevistado 3: “esse plano é importante e imprescindível para que Pelotas tenha o seu sistema Municipal de Cultura, e o sistema ele exige, faz algumas exigências, entre elas o plano, a existência do Conselho, as conferências, pra que tu estejas vinculado e seja reconhecido como sistema Municipal que vai se ligar ao sistema Estadual que vai se ligar o Sistema Nacional” (ENTREVISTADO 3). g Diante do exposto, fica evidente que o foco do Estado, neste momento, vai além dapublicação de editais, que delimita o número de contemplados e estabelece concorrência entre os atores culturais. Nesse período, o Estado parece investir na profissionalização e na descentralização, como categorias promotoras de uma multiplicidade de atores culturais independentes e responsáveis pelos resultados alcançados, ou seja, uma multiplicidade de indivíduos- empresa. Se o modo de atuação da Secult privilegia ações voltadas para uma proposta que aproxima os atores das ideias e praticas empresarias, a política de descentralização atua como uma ferramenta que as multiplica, que as dissemina. Situação idealizada pelo Estado neoliberal. E nessa multiplicidade de atores culturais, seguindo uma lógica predominantemente econômica, se encontra a materialidade da forma empresa. Isso porque esses atores agem segundo a formalização imposta pelos editais, e em busca de novas características de fora do contexto cultural, com base em pressupostos empresariais na busca por sobrevivência nesse ambiente. E, dessa forma, vão invadindo a cena cultural: a inovação, a formalização, a profissionalização, a lógica econômica, os cálculos utilitaristas, a busca por resultados, a melhoria contínua, a competitividade e a independência. E, então, ao se olhar para essas características, fica difícil saber do que se está falando, são atores culturais? São Trabalhadores culturais? Indivíduos-empresa? Ou são empresas? E aqui percebe-se o processo de disseminação da ideia de empresa, disposta nos mais diversos contextos. 4 CONSIDERAÇÕES FINAIS No primeiro período de formação da Secult, parece haver uma forma de atuação que se aproxima mais de um Estado keynesiano, com uma autonomia política e institucional, que sofre pouca interferência da economia de mercado e, consequentemente, da ideia de empresa. E quanto aos objetivos propostos pelo secretário, pode-se perceber uma convergência também do que é dito sobre o papel que a cultura representa e sua contribuição para o desenvolvimento, uma postura que converge a do estudo de Knopp et al (2010), no qual desenvolver é empoderar os indivíduos, ampliar a consciência sobre a sua condição, aumentar a sua capacidade de ação e sua liberdade substantiva, portanto, deve ser entendido como um projeto político de transformação social. No entanto, as ações do Estado, ao longo do tempo, começam a se consolidar em torno do ideário neoliberal. O Estado ainda ocupa um papel central, mas exerce funções diferentes. No regime de bem-estar social, o Estado toma para si a responsabilidade pelo desenvolvimento, enquanto que, no neoliberalismo, 91 SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 ele age para as instituições da esfera econômica, ainda que possa pareça estar tomando para si a incumbência. ele age para as instituições da esfera econômica, ainda que possa pareça estar tomando para si a incumbência. Através das posturas adotadas, percebe-se a presença de elementos neoliberais descritos por Foucault (2008), que estão alinhados ao processo de empresarização e caracterizam a atuação do Estado. O primeiro deles é a compreensão do trabalhador como responsável por melhorar sua condição, ou seja, como uma empresa para si mesmo. Essa compreensão estabelece a existência de uma estreita relação entre capital e competência, sendo capital o trabalhador capaz de gerar renda, a qual será relativamente maior, de acordo com a sua competência (FOUCAULT, 2008). Situação evidente com a publicação de editais e que é caracterizada como uma forma de democratizar o acesso à cultura. Por trás dos critérios impostos pelos editais, como ineditismo, qualificação do proponente, necessidade de gerar desdobramentos e autossustentabilidade, muitas manifestações podem estar fadadas ao desaparecimento ou à descaracterização. A arte, por exemplo, pode ser transformada em um produto disposto a atender uma demanda. Atores entram em contato e podem passar a incorporar os modelos tipicamente empresariais, como a busca por qualificação profissional, eficiência e resultados. 4 CONSIDERAÇÕES FINAIS p q ç p Nessa forma de conceber os editais, os candidatos são autorresponsabilizados pela aprovação do seu projeto, afinal de contas, o Estado diz deixar claro quais serão os critérios de avaliação mensurados. Essa aprovação permitirá o recebimento de determinada renda, de acordo com a relação existente entre o trabalhador e suas competências, cabendo a ele melhorá-las. Então, quando se diz que o Estado neoliberal atua centrado na forma empresa, não é em um modelo empresarial tradicional, é o incentivo para que os indivíduos assumam a forma empresa (FOUCAULT, 2008). E assumindo a forma empresa, esses indivíduos são incentivados a buscar qualificação profissional, seja pela disseminação de cursos de formação cultural pelos bairros da cidade, ou pelos editais que estabelecem currículo e qualificação profissional como critério de avaliação. No entanto, para que esses cursos de profissionalização obtenham sucesso, é necessário despertar nesses atores um ser empreendedor de si, para que reconheçam naqueles um investimento. E, como investidores, tornem-se empresários de si, responsáveis pela posição que ocupam e também pelo que conquistam. Logo se tornarão multiplicadores e reprodutores do comportamento empresarial, ações tão esperadas na lógica neoliberal. E é nessa perspectiva de crescimento, baseada no investimento em qualificação do capital humano, que Foucault (2008) nos fala que são executadas as políticas públicas atualmente, inclusive as culturais. Essa forma de lidar com os atores culturais, como se constituíssem sua própria empresa, insere-os em uma lógica econômica voltada para o mercado. E as relações concorrenciais aparecem como outro elemento desse Estado neoliberal. Naturais no ambiente empresarial, elas acabam por reconstituir uma série de valores morais e culturais. Esse modelo político é o que Foucault (2008) vai propor como a generalização da forma empresa no interior do corpo social, fazendo dos indivíduos uma espécie de empresa permanente e de empresas múltiplas. É, na economia do corpo social, organizado de acordo com as regras da economia de mercado, que as relações concorrenciais aparecem como um princípio de ordem. No entanto, esse princípio pode ser mais dissolvente do que unificante, tornando-se necessário que o Estado ocupe o seu lugar através da construção de leis que reforcem e mantenham o papel do Estado, como é o caso da lei de incentivo à cultura – Procultura. 4 CONSIDERAÇÕES FINAIS Essas relações concorrenciais fazem surgir, dentro da cena cultural, um ambiente próximo ao que está inserido a empresa, no qual ocorre concorrência entre empresas e, também, o estabelecimento de parcerias, a fim de se atingir um resultado. Então, percebe-se o Estado incentivando essas conexões, como forma de promover essas parcerias. Outra característica é a descentralização, na busca por uma multiplicidade de indivíduos atuando como se constituíssem, eles próprios, uma empresa. Aliada à ideia de profissionalização dos artistas, a descentralização parece ter a intenção de autonomizar e reproduzir indivíduos-empresa, ou seja, empresários independentes, inovadores, qualificados, com projetos autossustentáveis e competitivos, os futuros multiplicadores e reprodutores do comportamento empresarial. Ainda, evidenciou-se que algumas narrativas estão relacionadas a uma dimensão muito mais ampla, que engloba as esferas estadual e nacional, como é o caso da construção do Plano Municipal de Cultura. Esse é um projeto que teve início no ano de 2005 e guiou as ideias de constituir as conferências municipais de cultura ea criação dos editais de incentivo à cultura. Através da análise das posturas adotadas pelo Estado, representado aqui pelos agentes públicos que atuaram na Secult, pode-se perceber que, embora as primeiras ações não fossem permeadas por pressupostos da economia de mercado, com o passar dos anos, é notória a inserção da ideia de empresa no contexto cultural. É importante ressaltar que muitas das posturas adotadas referem-se a uma conjectura de esfera pública muito mais ampla do que o âmbito municipal, já que estão alinhadas a uma esfera nacional e estadual. a atenção o fato de a cultura e de as ações culturais concernentes ao Estado serem Chama a atenção o fato de a cultura e de as ações culturais concernentes ao SINERGIA, Rio Grande, v. 21, n. 2, p. 81-94, jul./dez. 2017 92 pautadas, majoritariamente, por uma lógica de distribuição de recursos. Isso representa forte relação e, até mesmo, um incentivo às ações promovidas e disseminadas no mercado cultural, espaço que, até então, parecia exclusivo dessa esfera. Ressalta-se que não se faz aqui nenhuma critica às ações de democratização do acesso aos recursos culturais, mas, sim,se demonstra que a maneira com que isso ocorreu representa forte alinhamento com o processo de empresarização. Ou seja, ainda que a intenção seja democratizar o acesso aos recursos públicos destinados a ações culturais, o que acabou ocorrendo foi a intensificação do processo de empresarização do campo. 4 CONSIDERAÇÕES FINAIS Talvez pelo fato de a empresa, na modernidade, ser apontada como a principal, quiçá a única solução. É É importante ressaltar que não se trata aqui de criticar comportamentos ou visões de mundo dos agentes públicos em relação à cultura, ou em relação ao papel do Estado frente à dimensão cultural. O que se está analisando, a partir das falas coletadas nas entrevistas, é a incorporação de um discurso amplo e disseminado na sociedade contemporânea: a centralidade da empresa seja como modelo e forma de organização. Essa pesquisa representa um trabalho inicial sobre o processo de empresarização, a partir de uma perspectiva foucaltiana, logo, teve um cunho exploratório com vistas a subsidiar pesquisas futuras relativas ao papel do Estado na disseminação da empresa, suas ideias e práticas, nos mais diversos contextos. Uma das principais limitações desse estudo refere-se à dificuldade de acesso aos dados secundários, principalmente documentos internos, já que muitos deles foram negados. Além disso, nesse momento,realizar entrevistas com outros atores da cena cultural pelotense, como empresários, artistas, participantes de projetos da secult, atividade que se pretende realizar em pesquisa futura. Ressalta-se aqui o trabalho de Martins, Tavares e Rodrigues (2016), o qual analisou a configuração do campo artístico pelotense sob a ótica dos atores culturais. Trata-se de pesquisas iniciais que permitem uma aproximação da discussão sobre o processo de empresarização no contexto cultural pelotense. 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https://openalex.org/W4211201764	https://www.med-alphabet.com/jour/article/download/1150/1126	Russian	null	Actual recommendations for multicomponent drug use in dermatology	Medicinskij alfavit	2,019	cc-by	3,746	Actual recommendations for multicomponent drug use in dermatology L. S. Kruglova, D. V. Fyodorov, A. G. Stenko, N. V. Gryazev, A. B. Yakovlev Central State Medical Academy of the Administrative Department of the President of Russia, National Scientific Centre for Children’s Health, People’s Friendship University of Russia, Institute of Plastic Surgery and Cosmetology; Moscow, Russia В помощь практическому врачу DOI: 10.33667/2078–5631–2019–2–26(401)-67–72 DOI: 10.33667/2078–5631–2019–2–26(401)-67–72 Резюме Многолетний опыт применения фиксированных комбинаций топических глюкокортикостероидов в дерматологии сегодня представляется обо- снованным не только теоретически (патогенез иммуновоспалительных и инфекционных дерматозов), но и с позиции оптимизации терапев- тических мероприятий у данной категории пациентов. С этой позиции инновационный комбинированный препарат Тетрадерм® можно расце- нивать как препарат выбора при дерматозах сочетанной этиологии. В его состав входят высокоэффективные компоненты, которые позволяют воздействовать на воспалительный компонент, инфекцию и трофику тканей в очагах поражения. Компоненты препарата показывают благо- приятный профиль антибактериальной и антимикотической активности. Высокопотентный кортикостероид позволяет эффективно купировать воспаление. Перспективы применения препарат имеет при различных дерматозах сочетанной этиологии (атопический дерматит, экзема), в клинических ситуациях, когда микозы или пиодермии имеют признаки экзематизации процесса, высыпания в крупных складках, пузырные дерматозы и дискератозы. Many years of experience in using fixed combinations of topical glucocorticosteroids in dermatology today seem to be justified not only theoretically (the pathogenesis of immuno-inflamma- tory and infectious dermatoses), but also from the perspective of optimizing therapeutic measures in this category of patients. From this position, the innovative combination drug Tetraderm® can be regarded as the drug of choice for dermatoses of com- bined etiology. The composition of the drug includes highly effective components that allow you to affect the inflammatory component, infection and trophic tissue in the lesions. The com- ponents of the drug show a favorable profile of antibacterial and antimycotic activity. Highly potent corticosteroid can effectively stop inflammation. The drug has prospects for use in various dermatoses of combined etiology (atopic dermatitis, eczema), in clinical situations when mycoses or pyoderma have signs of eczematization of the process, rashes in large folds, cystic dermatoses and diskeratoses. Key words: dermatoses of combined etiology, pyoderma, my- coses, combined preparation, Tetraderm®. Ключевые слова: дерматозы сочетанной этиологии, пиодермии, микозы, комбинированный препарат, Тетрадерм®. Прежде всего, композитные препара- ты с кортикостероидами используются при дерматозах смешанной природы, которые условно можно разделить на две группы: Актуальные рекомендации по применению многокомпонентного препарата в дерматологии Л. С. Круглова, д. м. н., проф., проректор по учебной работе, зав. кафедрой1 Д. В. Федоров, м. н. с. лаборатории патологии кожи2 А. Г. Стенько, д. м. н., член-корр. РАЕН, проф. кафедры3, зав. отделением4 Н. В. Грязева, к. м. н., доцент кафедры1 А. Б. Яковлев, к. м. н., доцент кафедры1 А. Г. Стенько, д. м. н., член-корр. РАЕН, проф. кафедры3, зав. отделением4 1Кафедра дерматовенерологии и косметологии ФГБУ ДПО «Центральная государственная медицинская академия» Управления делами Президента Российской Федерации, г. Москва 2 й й й р р р 2НИИ педиатрии ФГАУ «Национальный научно-практический центр здоровья детей» Минздрава России, г. Москва 3Кафедра эстетической медицины ФГАОУ ВО «Российский университет дружбы народов», г. Москва 4Отделение косметологии ОАО «Институт пластической хирургии и косметологии», г. Москва Актуальность проблемы образом: бактерии и (или) грибы явля- ются первичной этиологической при- чиной у 25 % больных дерматозами смешанной этиологии, присоединение вторичной микстинфекции отмечается у 56 % больных смешанными дермато- зами; бактерии и грибы — триггеры, провоцирующие обострение хрони- ческих дерматозов (19 % больных) [2]. образом: бактерии и (или) грибы явля- ются первичной этиологической при- чиной у 25 % больных дерматозами смешанной этиологии, присоединение вторичной микстинфекции отмечается у 56 % больных смешанными дермато- зами; бактерии и грибы — триггеры, провоцирующие обострение хрони- ческих дерматозов (19 % больных) [2]. Анализ современной литературы свидетельствует об отсутствии сни- жения заболеваемости пиодермиями, в то же время увеличивается этиоло- гическая значимость бактериальной и грибковой флоры в патогенезе хро- нических дерматозов [1]. Это связано прежде всего с тем, что в этиологии многих заболевания кожи бактерии и грибы выступают в качестве этиоло- гического агента — если не возбу- дителя, то очень важного фактора в патогенезе. Структура дерматозов сочетанной этиологии, по данным ряда авторов, выглядит следующим • стероидчувствительные дерма- тозы с потенциально возможным вторичным инфицированием; • инфекционные дерматозы с воз- можным развитием аллергической реакции или микстинфекции. Комбинированные топические препараты (кортикостероид + анти- биотик + антимикотик) занимают важное место в терапевтических меро- приятиях кожных болезней, при этом клинические ситуации, при которых они показаны, очень многообразны. Факторы, способствующие разви- тию вторично инфицированных со- стояний при стероидчувствительных дерматозах, включают: патогенез са- Медицинский алфавит № 26 / 2019, том № 2. Дерматология 6 E-mail: medalfavit@mail.ru В помощь практическому врачу Таблица 1 Терапевтический индекс (TIX) топических глюкокортикостероидных препаратов Терапевтический индекс (TIX) топических глюкокортикостероидных препаратов Препарат ГК ТРИ БМВ ГКБ КП МПА ММФ Вазоконстрикция 4 8 8 8 12 8 8 Сравнительная эффективность при АД 5 10 10 10 15 10 10 Общая оценка эффективности (Э) 9 18 18 18 27 18 18 Атрофия кожи 6 12 12 6 12 6 6 Влияние на системный баланс ГКС 2 4 2 2 4 2 2 Аллергический потенциал 1 1 1 1 1 1 1 Общая оценка безопасности (Б) 9 17 15 9 17 9 9 Терапевтический индекс (Э/Б) 1 1,06 1,2 2,0 1,5 2 2 Примечание: ГК — гидрокортизон, ТРИ — триамсинолон, БМВ — бетаметазона валерат, ГКБ — гидрокортизона бутират, КП — клобетазола пропионат, МПА — метилпреднизолона ацепонат, ММФ — мометазона фуроат. Примечание: ГК — гидрокортизон, ТРИ — триамсинолон, БМВ — бетаметазона валерат, ГКБ — гидрокортизона бутират, КП — клобетазола пропионат, МПА — метилпреднизолона ацепонат, ММФ — мометазона фуроат. ратом выбора в терапии таких стероид- чувствительных дерматозов, как атопи- ческий дерматит, себорейный дерматит, экзема и многие другие, обеспечивая клиническую эффективность терапии более чем в 90 % случаев независимо от диагноза [5]. Мометазона фуроат отличается высоким соотношением показателя «эффективность — безо- пасность» (табл. 1). Данные клини- ческих исследований свидетельствуют, что даже длительное использование препаратов с мометазоном фуроатом не приводит к атрофии кожи, развитию других нежелательных явлений, он обладает минимальным аллергизи- рующим потенциалом среди других ТГКС [6]. Препараты мометазона фу- роата включены в стандарты оказания медицинской помощи больным дерма- тозами и в клинические рекомендации РОДВиК [7–9]. мого дерматоза (нарушение барьерной функции, девиация во врожденной и адаптивной иммунной системах кожи, воспалительный процесс), при- менение иммунносупрессивных пре- паратов для терапии, входные ворота для инфекции (экскориации, ксероз и многое другое) [3]. тивность препарата как в отношении воспаления, так и инфекционного агента, и безопасность. тивность препарата как в отношении воспаления, так и инфекционного агента, и безопасность. Таблица 3 ные о чувствительности к эконазолу грибов рода Candid Данные о чувствительности к эконазолу грибов рода Candida Д у у р р Материал исследования Чувствительность штаммов к Эконазолу 439 больных ВВК, выделено 593 штамма грибов Candida: C. albicans (70,9 %), C. glabrata (18,9 %), C. parapsilosis (5,1 %) и др. [21] Чувствительность отмечалась у 94,3–98,5 % штаммов; (MIC ≤ 1 мг/л) 93 штамма грибов Candida: C. albicans (62,4 %), C. glabrata (26,9 %) [22] Все изоляты были чувствительны: 93,5 и 6,5 % были дозозависимыми 200 изолятов грибов Candida из очагов ВВК и с кожи [23] Для изолятов C. albicans: 0,015–0,250 мг/л; для изолятов C. non-albicans: 0,5–80; 1,0–4,0 и 0,12–4,00 мг/л 100 изолятов грибов Candida из очагов ВВК и с кожи [24] Все изоляты C. albicans были чувствительны: MIC 0,016–16,000 мг/л Чувствительность штаммов к Эконазолу Чувствительность отмечалась у 94,3–98,5 % штаммов; (MIC ≤ 1 мг/л) Все изоляты были чувствительны: 93,5 и 6,5 % были дозозависимыми Для изолятов C. albicans: 0,015–0,250 мг/л; для изолятов C. non-albicans: 0,5–80; 1,0–4,0 и 0,12–4,00 мг/л Все изоляты C. albicans были чувствительны: MIC 0,016–16,000 мг/л Материал исследования 439 больных ВВК, выделено 593 штамма грибов Candida: C. albicans (70,9 %), C. glabrata (18,9 %), C. parapsilosis (5,1 %) и др. [21] 93 штамма грибов Candida: C. albicans (62,4 %), C. glabrata (26,9 %) [22] 200 изолятов грибов Candida из очагов ВВК и с кожи [23] 100 изолятов грибов Candida из очагов ВВК и с кожи [24] Материал исследования 439 больных ВВК, выделено 593 штамма грибов Candida: C. albicans (70,9 %), C. glabrata (18,9 %), C. parapsilosis (5,1 %) и др. [21] 93 штамма грибов Candida: C. albicans (62,4 %), C. glabrata (26,9 %) [22] 200 изолятов грибов Candida из очагов ВВК и с кожи [23] 100 изолятов грибов Candida из очагов ВВК и с кожи [24] ( ) Наиболее частыми возбудите- лями дерматофитий являются пато- генные грибы Trichophyton rubrum (90 %) и Trichophyton mentagrophytes, var. Interdigitale, реже эти заболевания вызываются Epidermophyton floccosum и грибами рода Candida. При вторично инфицированных дерматозах наибо- лее часто встречаются грибы рода Candida и Malassezia. Поэтому при эмпирическом назначении препаратов с антимикотической активностью не- обходимо, чтобы препарат был эффек- тивен в отношении широкого спектра грибов и, в частности в отношении Candida (табл. 3). Эконазол — про- тивогрибковый препарат из группы азолов, синтетическое производное имидазола с преимущественно фун- гистатическим действием. Эконазол действует на дерматофитов, Candida spp., Malassezia spp., некоторые грам- положительные бактерии. Комбинированные с кортикостероидами препараты Тетрадерм® (Tetraderm) — крем для наружного применения. АТХ- классификация: D 07XC 03 мометазон в комбинации с другими препарата- ми. Фармакологическая группа: анти- биотик-аминогликозид, декспантенол, кортикостероид для местного приме- нения, противогрибковое средство. На сегодняшний день аналоги данного препарата в мире отсутствуют, более того, эти активные вещества в различ- ных сочетаниях никогда не входили в состав фиксированных комбинаций. В свою очередь пиодермии и ми- козы в определенных локализациях (складки, места с повышенной склон- ностью к мацерации) у лиц с сопутству- ющей соматической патологией (сахар- ный диабет, сосудистые заболевания, метаболические нарушения), а также на фоне нерационально подобранной терапии (несоответствие лекарствен- ной формы препарата и локализации или остроты процесса), при сенсиби- лизации к компонентам топического средства могут сопровождаться ал- лергическими реакциям и приводить к развитию патологического процесса уже сочетанной этиологии [4]. Активность препарата Тетрадерм® обусловлена фармакологическими свойствами компонентов, входящих в его состав: мометазона фуроат ока- зывает противовоспалительное, проти- возудное, антиэкссудативное антипро- лиферативное действие, стимулирует апоптоз активированных клеток; ген- тамицина сульфат оказывает противо- воспалительное, противомикробное действие, эконазола нитрат — проти- вовоспалительное, противогрибковое (фунгицидное) действие; дексапан- тенол — противовоспалительное, ранозаживляющее, регенеративное действие. Такая комбинация, безус- ловно, является инновационной и пер- спективной в плане применения при широком спектре дерматозов. В отношении антибактериальной и антимикотической составляющих основными факторами эффективности лекарственных препаратов являются чувствительность к ним микроорга- низмов. На сегодняшний день основ- ным патогеном при вторично инфи- цированных дерматозах и пиодермиях считается золотистый стафилококк S. aureus (61,4 %), реже встречаются дру- гие возбудители, например стрепто- кокк S. pyogenes (25,7 %) [10]. Интерес исследователей к стафилококкам и прежде всего к S aureus при разных дерматозах обусловлен существующей проблемой резистентности и сверхре- зистентности. Реальностью в дермато- логии стали метициллин-резистентные (MRSA) и полирезистентные штамы стафилококка [11]. Наиболее часто показаниями к назначению комбинированных кортикостероидов являются следу- ющие нозологии: экзема, атопиче- ский дерматит, пиодермии, микозы, а также смешанные поражения или осложненные вторичной инфекцией воспалительные и аллергические за- болевания. В большинстве случаев практический врач назначает комби- нированный препарат эмпирически, лишь предполагая причину дерматоза, так как посевы на микрофлору и чув- ствительность к антибиотикам и ан- тимикотикам занимают длительное время и не всегда помогают выявить возбудителя. Поэтому очень важными аспектами являются высокая эффек- Мометазона фуроат — топический стероид с доказанной эффективностью и безопасностью, он оказывает патоге- нетическое действие и является препа- Медицинский алфавит № 26 / 2019, том № 2. Дерматология E-mail: medalfavit@mail.ru В помощь практическому врачу В состав Тетрадерма® входит аминогликозид гентамицин. Таблица 3 Эконазола нитрат применяется как средство местной терапии дерматофитии и кандидоза кожи, в том числе ос- ложненных вторичной бактериаль- ной инфекцией, разноцветного лишая, эритразмы, а также при вагинальном кандидозе и отомикозах [20]. Хейли-Хейли), у 4 (5,3 %) — аллер- гический дерматит, осложненный вторичной инфекцией, у 4 (5,3 %) — дискератоз Дарье, у 3 (4,0 %) пациен- тов — пиодермия с экссудацией. Вне зависимости от нозологии пациенты получали топическую терапию в виде крема Тетрадерм® два раза в день на протяжении до 4 недель (дли- тельность курса определялась кли- нической картиной и была различной в группах пациентов). Если основное заболевание требовало системной те- рапии, пациенты получали системное лечение в соответствии с клинически- ми рекомендациями. ность при различных заболеваниях кожи подтверждена в ходе более чем 100 клинических исследований [25, 26]. Основные показания для назначения дексапантенола постоянно расширя- ются и на сегодняшний день включают атопический дерматит, пеленочный дерматит, экзему, а также дерматозы с нарушением барьерной функции с целью восстановления и улучшения репаративных процессов [25, 26]. Комбинированные с кортикостероидами препараты При эмпирическом назначении антибак- териальных препаратов врач должен обладать актуальной информацией о чувствительности основных пато- генов, чтобы сделать правильный выбор. В исследованиях последних лет (2014, 2016) была показана вы- сокая чувствительность к гентами- цину различных бактерий, в том числе и S. аureus [12, 13]. Данные о чувствительности к гентамицину штаммов S. аureus показывают, что на протяжении последних десятиле- тий отсутствует отрицательная дина- мика в отношении резистентности, в частности S. aureus (табл. 2). Таблица 2 Данные о чувствительности к гентамицину штаммов золотистого стафилококка Данные о чувствительности к гентамицину штаммов золотистого стафилококка Материал для тестирования Чувствительные штаммы к гентамицину, % 1 152 штамма S. aureus, 7 регионов Франции [14] 89,0 % 898 штаммов S. aureus, г. Москва и Санкт-Петербург [15] 81,0–94,0 % 879 штаммов S. aureus, 12 регионов РФ [16] 69,3 % 294 штамма S. aureus, Литва [17] 96,4 % MIC от 0,25 до 2,00 мг/л 480 штаммов S. aureus, 7 регионов Франции [18] 95,5 % 97 штаммов S. aureus, Китай [19] 93,8 % Таблица 3 Данные о чувствительности к эконазолу грибов рода Candida В помощь практическому врачу В помощь практическому врачу Рисунок 1. Динамика показателей индекса ДИШС. Результаты исследования Под наблюдением находилось 75 пациентов с дерматозами сочетанной этиологии. Среди них 41 мужчина и 34 женщины, возраст варьиро- вал в широких пределах (от 18 лет до 79 лет) и в среднем составил 39,2 ± 5,8 года. Оценка эффективности проводи- лась в соответствии с дерматологи- ческим индексом ДИШС: эритема, папулы, пустулы, отек по трехбалль- ной шкале, где 0 баллов — элементы сыпи отсутствуют; 1 балл — слабо выражены, единичные элементы; 2 балла — умеренно выражены, эле- менты; 3 балла — сильно выражены, множественные элементы. У 21 (28 %) пациента была диагно- стирована экзема, у 18 (24 %) — ато- пический дерматит, у 9 (12 %) — ми- козы гладкой кожи с экзематизацией (эпидермофития паховой области, кандидоз кожи, разноцветный лишай), у 8 (10,7 %) — себорейный дерматит, у 8 (10,7 %) — пузырные дерматозы с признаками вторичного инфициро- вания (пузырчатка, буллезный пемфи- гоид, доброкачественная пузырчатка Еще одно действующее вещество препарата Тетрадерм® — декспанте- нол 5 % — провитамин пантотеновой кислоты (B 5), необходимый компонент метаболизма. Клинически доказаны регенерирующее, метаболическое и умеренное противовоспалительное действия декспантенола и высокая его безопасность (GRAS-status: может быть добавлен в пищу), а эффектив- После терапии во всех группах от- мечалась положительная динамика в отношении клинических симптомов, при этом динамика составила более 90 % (см. рис.). Медицинский алфавит № 26 / 2019, том № 2. Дерматология E-mail: medalfavit@mail.ru Заключение Многолетний опыт применения фиксированных комбинаций топиче- ских глюкокортикостероидов в дер- матологии сегодня представляется обоснованным не только теоретиче- ски (патогенез иммуновоспалитель- ных и инфекционных дерматозов), но и с позиции оптимизации тера- певтических мероприятий у данной категории пациентов. Понимание ведущей роли таких патогенов, как Staphylococcus aureus, Candida albicans и Malassezia spp. (в том числе в виде микст-инфекции), не только при ин- фекционных патологиях, но и в пато- генезе атопического дерматита и эк- земы, в качестве факторов вторично инфицированных состояний при раз- личных дерматозах делает обоснован- ным назначение многокомпонентных топических средств. Важным аспектом наружной терапии кожных болезней является восстановление барьерной функции кожи, чему может способ- ствовать усиление трофики как след- ствие действия такого компонента, как декспантенол. С этой позиции иннова- ционный комбинированный препарат Тетрадерм® можно расценивать как потенциально универсальный для ку- пирования воспаления, элиминации патогена и восстановления барьерной функции кожи. фу Новый отечественный комби- нированный препарат Тетрадерм® является актуальным топическим средством для широкого примене- ния в дерматологической практике. В его состав входят высокоэффектив- ные компоненты, которые позволяют воздействовать на воспалительный компонент, инфекцию и трофику тка- ней в очагах поражения. Компоненты препарата показывают благоприят- ный профиль антибактериальной и антимикотической активности. Высокопотентный кортикостероид позволяет эффективно купировать воспаление. Перспективы применения препа- рат имеет при различных дерматозах сочетанной этиологии, в клинических ситуациях, когда микозы и пиодер- мии имеют признаки экзематизации процесса, высыпаниях в крупных складках, пузырных дерматозах и дискератозах. Рисунок 1. Динамика показателей индекса ДИШС. Медицинский алфавит № 26 / 2019, том № 2. Дерматология E-mail: medalfavit@mail.ru Список литературы 1. Масюкова С. А., Гладько В. В., Устинов М. В. и др. Бактериальные инфекции кожи и их значение в клинической практике дермато- лога // Consilium medicum. 2004. — Том 6. — № 3. — С. 180–185. 19. Yılmaz E. S., Aslantaş Ö. Antimicrobial resistance and underlying mechanisms in Staphylococcus aureus isolates. Asian Pacific Journal of Tropical Medicine 2017; 10 (11): 1059–1064. 10. Белькова Ю. А. Фармакоэпидемиологические и микробиологические аспекты оптимизации лечения инфекций кожи и мягких тканей в ам- булаторных условиях. Смоленск. — 2004. — Ав- тореф. дисс. канд. мед. наук. — 24 с. 2. Белоусова Т. А., Горячкина М. В. Алгоритм на- ружной терапии дерматозов сочетанной этиологии. Вест дерматол и венерол. 2011; 5: 138–144. 20. Сергеев А. Ю. и др. Доказательный опыт тера- пии дерматомикозов сертаконазолом // Им- мунопатология, аллергология, инфектология. — 2015. — Т. 4. — С. 98–110. 11. 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Screening Antimicrobial Susceptibility of Gentamicin, Vancomycin, Azi- thromycin, Chloramphenicol and Cefotaxime Against Selected Gram Positive and Gram Neg- ative Bacteria. International Journal of Pharma Research and Health Sciences. 2014; (4): 324–331. 22. Mahmoudabadi A. Z. Antifungal Susceptibility of Candida Species Isolated From Candiduria. Jundishapur Journal of Microbiology 6 (1): 24–28. 5. Короткий Н. Г., Тихомиров А. А., Гамаю- нов Б. Н. Сравнительная эффективность Момата (мометазона фуроата) и гидро- кортизона 17–бутирата в наружной те- рапии атопического дерматита у детей. Русский медицинский жернал. — 2008. — С. 1183–1186. 23. Choukri F., Benderdouche M., Sednaoui P. In vitro susceptibility profile of 200 recent clinical isolates of Candida spp. to topical antifungal treatments of vulvovaginal candidiasis, the imid- azoles and nystatin agents. Journal de Mycologie Médicale. — 2014. Principles of diagnostics and therapy of anogenital pruritus in women Yu. N. Perlamutrov, Т. V. Vаsilеnkо, К. B. Olkhovskaia Moscow State University of Medicine and Dentistry n. a. A. I. Evdokimov, Moscow, Russia В помощь практическому врачу В помощь практическому врачу 9. Chi С. С. et al. Updated evidence-based (S 2e) European Dermatology Forum guideline on topical corticosteroids in pregnancy. JEADV 2017; 31: 761–773. 18. Bernard P. et al. Antibiotic susceptibility of Staph- ylococcus aureus strains responsible for com- munity-acquired skin infections. Ann Dermatol Venereol. 2008 Jan; 135 (1): 13–9. Список литературы Список литературы Volume 24, Issue 4, P. 303–307. 14. Moorhouse E., Fenelon L., Hone R., et al. Staphylococ- cus aureus sensitivity to various antibiotics a national survey in Ireland 1993. Ir J Med Sci. 1996; 65 (1): 40–43. 15. Сидоренко C. B., Резван С. П., Грудинина С. А. и др. Результаты многоцентрового исследова- ния чувствительности стафилококков к анти- биотикам в Москве и Санкт-Петербурге. 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Gailiene Staphylococcus aureus resistance to antibiotics and spread of phage types Medicina (Kaunas), 42 (2006), pp. 332–339. 8. Gual A, Pau-Charles I, Abeck D. Topical Cortico- steroids in Dermatology: From Chemical Devel- opment to Galenic Innovation and Therapeutic Trends. 2015; J Clin Exp Dermatol Res 6: 269–72. 26. Proksch E. et al. Topical use of dexpanthenol: a 70th anniversary article. Journal of Dermatolog- ical Treatment, 2017; 28 (8): 766–773. Trends. 2015; J Clin Exp Dermatol Res 6: 269–72. Для цитирования. Круглова Л. С., Федоров Д. В., Стенько А. Г., Грязева Н. В., Яковлев А. Б. Актуальные рекомендации по применению много- компонентного препарата в дерматологии // Медицинский алфавит. Серия «Дерматология». — 2019. — Т. 2. — 26 (401). — С. 67–72. Для цитирования. Круглова Л. С., Федоров Д. В., Стенько А. Г., Грязева Н. В., Яковлев А. Б. Актуальные рекомендации компонентного препарата в дерматологии // Медицинский алфавит. Серия «Дерматология». — 2019. — Т. 2. — 26 ( Принципы диагностики и терапии аногенительного зуда у женщин Ю. Н. Перламутров, д. м. н., проф., зав. кафедрой, заслуженный врач России К. Б. Ольховская, к. м. н., доцент Кафедра кожных и венерических болезней ФГБОУ ВО «Московский государственный медико- стоматологический университет имени А. И. Евдокимова» Минздрава России, г. Москва Summary Summary Anogenital itching is a serious problem for most women at one or another period of life. The multifactorial nature of dermatoses with localization in the vulva and anus, the lack of knowledge of the pathophysiology of itching, in principle, determine the problem of itching in the genital area as a relevant medical problem. Conducting a rational differential diagnosis and the use of modern treatment that can quickly jugulate a symptom will at the present stage increase the effectiveness of specialized medical care for this category of patients. Key words: anogenital pruritus, etiology, pathophysiology, differential diagnosis, therapy. Резюме Зуд аногенитальной области является серьезной проблемой преимуще- ственно женщин в тот или иной период жизни. Мультифакториальность дерматозов с локализацией в области вульвы и ануса, отсутствие изученности патофизиологии зуда в принципе определяют данную проблему как актуальную. Проведение рациональной дифференци- альной диагностики и использование современных средств, способных быстро купировать симптом, позволяют повысить эффективность специализированной медицинской помощи данной категории больных. Ключевые слова: аногентальный зуд, этиология, патофизиология, диф- ференциальная диагностика, терапия. Ключевые слова: аногентальный зуд, этиология, патофизиология, диф- ференциальная диагностика, терапия. Медицинский алфавит № 26 / 2019, том № 2. Дерматология Медицинский алфавит № 26 / 2019, том № 2. Дерматология E-mail: medalfavit@mail.ru
https://openalex.org/W1933489380	https://njvet.ep.liu.se/article/download/3202/2534	English	null	Maintenance or change - an analytical tool for studies of the Work-Integrated Learning in teacher education	Nordic journal of vocational education and training	2,014	cc-by	9,015	Abstract In Sweden, as in many other countries, work-integrated learning is an emphasized part of teacher education. In parallel with this emphasis, the conflict often emerging in students' meeting with the profession is well known from previous research. In spite there seem to be few analytical tools for capturing the characteristics of work-integrated learning. The aim of this article is to construct, operationalize and try out an analytical tool to be used in connection with studies of the character of work-integrated learning. For the construction of the tool we rely on the conceptual pair of horizontal and vertical discourse, collected from Basil Bernstein's theoretical frame. The horizontal discourse connects to an informal, everyday context whereas the vertical discourse is formal and school-oriented. In the article we divide these concepts into certain aspects related to work-integrated learning. We also turn to empirical material where student teachers were interviewed about their experiences, mainly from tutorial guidance. Our conclusion is that the analytical tool could serve as a useful instrument for analyzing such phenomena. Maintenance or change - an analytical tool for studies of the Work-Integrated Learning in teacher education Jörgen Dimenäs, PhD, University of Gothenburg; Anita Norlund, PhD, University of Borås NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article Vol. 4 2014 Nordic Journal of Vocational Education and Training f g become the subject of further discussion between tutor and student. Ahlström (2012) also draws attention to this discrepancy and argues that Swedish teacher education today do efforts to link academia with teachers' professional activities. Secondly, there are numerous written texts that have focused on tutoring problems (Lauvås and Handal, 2001, Hegender 2010, Ahlström 2012), relevant to our article since we rely on the assumption that the character of the work-integrated learning is heavily dependent on tutorial phenomena. Ahlström (2012), for instance, has drawn the conclusion that some progress could be seen in this area but he argues that the primary focus in tutoring seems to be on procedural knowledge. A similar result is found in Hegender’s (2010, 2012) studies, which describe how tutors primarily focus on knowledge and skills in their communication with students. Hegender’s conclusion is that assessment mainly emphasizes procedures as well as social, emotional and caring aspects of teachers' work. The communication and dialogue with the student is often based on their own experiences and rarely has any connection to educational research. Wedin, Hultman and Schultz (2012) note that although tutors are educated in a supervisor training course they still focus on personal characteristics of the local school context in dialogues with the students. As already mentioned, we particularly include the phenomenon of tutorial in work- integrated learning and there are a number of studies highlighting the tutoring situation in such, i. e. what happens in the relationship between the supervisor and the student teacher. For instance, Hultman, Schultz and Post (2011) draw the conclusion of such supervision; the exchange of learning is based on the supervisor's observations and the subsequent talks seem to be characterized by elucidations, and by alternative ways to understand situations in the classroom. The authors believe that the results of their study are similar to several other studies pointing to a belief that "you learn to swim by being thrown directly into the water" (p. 133). The meetings that occur between supervisor and student teacher mean that different situations and dilemmas are discussed. Although the awareness of these dilemmas the research field of work-integrated learning has been characterized as undertheorized, and the same goes for tutorial as such. An attempt to theorize the tutoring situation in particular is found in Halse and Malfroy (2010) who want to challenge the lack of theorization in tutoring. NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article Introduction The present study focuses on the practical part of teacher education, from now on referred to as work-integrated learning (WIL), and offers an analytical tool for how to capture the nature of this learning. Work-integrated learning is a phenomenon of particular concern for several reasons. The work-integrated learning periods are usually highly valued by student teachers and, considered an important element for their future occupation. Moreover, its importance is noticeable from the fact that work-integrated learning makes up as much as a total of at least one semester in most Swedish teacher education programs. Finally, it is emphasized also by the government, which has clearly stated that work-integrated learning should help teacher students to relate content area matters both to the theoretical content of higher education and to practical teaching (1999/2000: 135). Another reason for paying attention to work-integrated learning is the fact that it is not without its problems. There are several studies revealing dilemmas connected to the encounter between teacher education and elements of work- integrated learning. One dilemma connects to how students relate what we want to call the academy on the one hand and the teaching profession on the other, a phenomenon usually referred to as the dyad of teacher education theory and practice (Shulman, 1998, Bloomfield, Taylor and Maxwell, 2004; Gustavsson, 2008; Dimenäs, 2010). As a contrast to these aspects, Eriksson (2009) shows in her study that there are attempts in teacher education to link academia with the profession during the students' practical parts of teacher education. These links stem from tasks undertaken during ordinary university periods and will subsequently 1 NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Theory Vol. 4 2014 Our study should be seen as an attempt to take the present discussion of the work- integrated learning of teacher education a step further, and it is done so by a focus on a theoretical conceptual pair. More specifically, we rely on the conceptual pair of two knowledge forms i. e. horizontal and vertical discourse. The pair is collected from Basil Bernstein's pedagogic code theory (see f. ex. Bernstein, 1999). In essence we have to do with the ‘local’ and ‘everyday’ on the one hand and the ‘official’ and ‘school-oriented’ on the other. It is important to state that the two discourses should not be seen as dichotomous. As a matter of fact Bernstein has introduced the new concepts in order to meet what he saw as a simplified and dichotomized view between the two, i. e. official school and local everyday. He experienced that there was the need for a language that helped differentiate between the two knowledge forms as well as within them. As far as the two discourses are concerned and the differences between them, the vertical form is characterized by decontextualization, specialization and formality. It is general, abstract, distant (Breier, 2004) as well as unfamiliar (Bourne, 2004). It is distributed in a particular way; since it is characterized by specialized language, knowledge is made accessible for the students (the acquirers) in a certain recontextualization process. This means that knowledge from the academia is relayed to teacher education and to the individual student, and implies a process where the student gets so familiar with the new knowledge that it may be consciously and intentionally transferred from one situation to the other. Ultimately, this transmission aims at competences through which knowledge may be handled individually by the student. Every individual student is supposed to get access to knowledge and to be able to transfer knowledge from one situation to the other without being dependent on the context. In turn, a horizontal discourse is context-dependent, characterized by everydayness, spontaneity and informality, and, according to Bourne (2004), includes the familiar and the concrete. A horizontal form is often played out in a context of family or friends and it cannot easily be distanced from its context. It is most likely to occur in situations that are familiar and recognizable and the acquirer is probably supported by others in such situations. Nordic Journal of Vocational Education and Training Previous analytical tool is offered as an attempt to facilitate and capture the nature and understanding of tutoring during work-integrated learning. Although we are fully aware that previous traditions/research has paid some attention to theoretical and analytical aspects, we argue that there is a need for an adequate tool that takes into account teacher education with its particular tradition (see below). Norlund (2013) operationalized the tool in connection to a different content and we find it important to try out its potential regarding work-integrated learning. Given these assumptions the aim of this article is to construct, operationalize and try out an analytic tool to be used in connection to studies of the character of work-integrated learning. 2 NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Nordic Journal of Vocational Education and Training Vol. 4 2014 an activity of a vertical character. Our exposition to come relies on this assumption and the conclusions to be drawn will be based in alignment with this. When, in turn, we have to deal with differentiation within the vertical discourse there are, according to Bernstein, different character of vertically oriented knowledge depending on what academic discipline the knowledge is based in. In other words, different academic disciplines have different knowledge structures. For instance, what belongs to the natural sciences has a certain hierarchic structure whereas humanities fall within a horizontal structure. This fact is of immediate interest for this article. Work-integrated learning within teacher education has yet another origin than a traditional academic education. We can go back to the early 1900s to find special teacher seminars in Sweden with a close connection to the profession. During the latter part of the 1900s, teacher training became part of an academic professional training. Here, the basic idea was to involve a university research tradition including its scientific basis in order to develop teacher education, and, in this way get more qualified teaching staff. This, in turn, was assumed to lead to improved teaching approaches in school. Such a description applies both to a Swedish and an international western perspective and has often been discussed in terms of a conflicted and dichotomized picture of teacher education, sometimes leading to difficulties for students to understand (Grossman and McDonald, 2008; Grossman, Hammerness and McDonald, 2009; Dimenäs, 2010). Also this picture adds to the needs we attempt to meet in this article. Before moving on, there are yet a couple of clarifications to do. First, an important fact in line with the theory is that the gate should be opened to the vertical discourse and it should not be considered appropriate to exercise teaching or tutoring by being oriented towards the horizontal discourse. Specialized knowledge as in the vertical discourse, it is believed, is needed in relation to perform good teaching. Teacher education has the responsibility of opening to the vertical discourse and work-integrated learning periods are in this respect considered an important tool, i. e. by giving flesh and blood to the theoretical parts and possibly making this process easier. The theory also relates to the future teachers’ possibilities of analyzing educational matters in particular and the position of education in society in general. NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Theory Another characteristic of the horizontal discourse is that everyone has access to this kind of knowledge. In other words it is kind of a collective property. In contrast to the vertical knowledge form, characteristic is that the acquirer does not experience a conscious progression between different learning situations and knowledge is not easily transferred from one situation to another. This fact is expressed as if the horizontal knowledge form is distributed segmentally. Another important difference between the two is the ways through which knowledge is evaluated. Normally, activities realized within a horizontal discourse, according to Bernstein’s starting points, are not evaluated in the same systematic way as in the vertical. Only in the formal situation (Bernstein, 2000) are there institutionally drawn-up criteria, in this case, for what it means being a good teacher. Thus, a further crucial point dividing the two discourses is the fact that the vertical discourse relates to a public and professional institution, in this case to teacher colleges, with the authority to evaluate, in this case teacher students. Due to this, work-integrated learning in teacher education is inevitably 3 NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article The construction of the analytical tool The analytical tool to be presented is presented in a table (see table 1) where the two discourses vertical and horizontal are given a column respectively. Unfortunately, the table version risks presenting the two discourses as dichotomous. As mentioned before, the non- dichotomous feature is fundamental for Bernstein's theory (cf. Moore and Muller, 2002). So, when, and if, the table implies that everyday activities are carried out without competence or specialization, this should be considered a misconception. Activities taking place in an everyday context are not detached from competence. However, it can be assumed that they are often played out with a lower degree of consciousness, sometimes called “tacit knowledge” (Zhang and Han, 2008). By using the table version, we follow Bernstein’s own example since he has himself presented the most important differences of the conceptual pair (Bernstein, 1999, p. 162) i.e. those related in the previous section, in a table. In addition, we 4 Nordic Journal of Vocational Education and Training Selection Vol. 4 2014 Our article should not only be seen as empirically grounded, although we make use of some empirical material. However, in order to try out the analytical tool and to illustrate an analysis we have chosen to work with empirical data. These data are based on a study of a qualitative design. The data collection method used is semi-structured (Kvale, 1996) interviews with students, carried out both individually and in groups. The sample consists of 15 student teachers who had recently completed their last period of work-integrated learning in preschool through to secondary school. The interview study focused how the student teachers had perceived the communication with their tutors, what advice had been prioritized and what feedback and feed forward they had received from their supervisors. It will be shown that the students’ overall experiences, from their work-integrated learning, were also paid attention. Our empirical material involves statements made by the students, meaning that it is only the students’ experiences that are given priority in the analysis to follow, not the factual circumstances. Data were processed in relation to the analytical model presented above (table 1). The analytic tool should be seen as an attempt of offering a useful apparatus for researchers who are interested in analyses of various kinds, such as observations of work- integrated learning or interviews with supervisors. Vol. 4 2014 f g stick to the visual form of a table for reasons of readability and symmetry. When constructing the analytical tool for studies of the character of work-integrated learning, our focus has been on certain aspects. The selection of these aspects is based on a combination of principles. First, it includes certain differences customarily emphasized in the Bernsteinian theory (see eg. Bernstein, 1990, 2000; Norlund, 2009). Second, it takes into consideration a previous study where the tool was constructed and tried out in relation to a completely different content, i. e. classroom debates among secondary school pupils (Norlund, 2013). A third principle has been to include what might be considered particularly relevant in connection to work integrated learning. Our intention with the final combination is to get close to the principles of “pedagogic transmission/acquisition” given great attention by Bernstein (1999). When we ourselves define transmission and acquisition we partially rely on an overarching division, common in curricular theory including selection, organization and evaluation. Table 1. Overview of the contents and characteristics of work-integrated learning realized in a vertical or a horizontal discourse (Freely after Bernstein, 1999). Table 1. Overview of the contents and characteristics of work-integrated learning realized in a vertical or a horizontal discourse (Freely after Bernstein, 1999). a vertical or a horizontal discourse (Freely after Bernstein, 1999). 5 NordYrk - ISSN 2242-458X – Article The application of the analytical tool In this section, we will put the tool in motion in order to facilitate for the reader to judge its usefulness. We will also make some comments in connection to the empirical material to illustrate how such may be formulated. We treat the aspects from table 1 respectively, which possibly will illustrate the analytic definitions by the use of our empirical data. Table 2. The aspect of information material Table 2. The aspect of information material Table 2. The aspect of information material Table 2. The aspect of information material Table 2. The aspect of information material Very little use of texts or other public/professional material is revealed in our material. This circumstance operates within a horizontal discourse and illustrates how the analytical tool can be used. When a text is mentioned it is still operating in a horizontal discourse, since the tutor seems to be unacquainted with the curricular documents: 6 NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Vol. 4 2014 When I related [my planning] to the curricular learning targets … “Oh, my God, how good” [tutor’s exclamation], to her it was so awfully new, that it was no matter of course to her. (Jill) What Jill refers to is a tutor who is not accustomed to turn to the curricular documents. Given the assumption that tutoring activities benefit from being shaped by the use of professional texts, the teacher student Jill’s experience is worth considering. Table 3. The aspect of topics and contents involved Table 3. The aspect of topics and contents involved Table 3. The aspect of topics and contents involved We are now moving to the next aspect of the analytical tool, the aspect connected to the potential to fracture hierarchies. This aspect is in fact one of the key elements of the analysis made and marks the connection between the analytic tool and its sociological tradition, although all vertical orientations do so in a more implicit way. First, we would like to share an utterance from one of the tutors referring to teacher students. It connects to a teacher’s everyday conditions: It is not easy to work in this district. But, how do you keep going? Some days, he just ... "what should I do?" Just grab things over again, start over. (Amelia) This statement, characterized as it is by resignation, indicates a teacher who is reluctant to take action with regards to a difficult professional situation. As such, it is an example in line with a horizontal discourse, where students to a lesser extent might be encouraged to question teachers’ positions in general. A parallel and alternative interpretation is that the above quotation signifies a teacher who does not give up supporting his pupils. It also enables the questioning of society and of underprivileged positions for pupils as well as teachers. Table 4. Vol. 4 2014 The analytic tool in use in this article places interest in the parts of the professional duties that are in focus during tutoring conversations (or recalled situations). Naturally, the teacher profession draws on a wide range of aspects, and it would not be realistic to expect that all of them appear in an interview of the kind that is carried out in this study. Still, there are some phenomena to which we pay special attention. For a presentation of these we rely on a triad (Norlund et al., 2012), in which we find three core elements (that cannot be separated from each other). First, it has to do with the transmission of values and the responsibility to encourage certain pupil identities and behaviour. Second, it has to do with general skills and competences. Third, we have the teaching of content area matters that belong to school subjects. Let us start in the latter. According to our interviews, this part seems at times to have been neglected. One of the teacher students interviewed Krister, says that, "I have not received any comments regarding the content in my teaching”. He specifies his comment: My supervisor is good at discussing everything that happens outside the classroom, outside the school subjects. It’s her focus. Those are the discussions that take place at school. What has been planned is put off. (Krister) In fact, the material only reveals a few examples of tutoring situations that have touched on subject-specific content. Jytte’s supervisor even signals that such content should be seen as unproblematic: My supervisor even thought that those things that have do with lessons is …, “that is something that you fix easily, it's everything around that is the hard part”. (Jytte) My supervisor even thought that those things that have do with lessons is …, “that is something that you fix easily, it's everything around that is the hard part”. Table 2. The aspect of information material The aspect of elements of the profession Table 4. The aspect of elements of the profession Table 4. The aspect of elements of the profession 7 NordYrk - ISSN 2242-458X – Article Vol. 4 2014 f g Another illustrative example is Axel who graded tests during his work integrated learning but did not discuss the grading activity, neither in general nor in particular. This possibly reveals a tutor who puts great faith in the student since Axel is in the end of his education. Interpreted in such a way, the tutor’s actions certainly make sense. However, an immediately apparent problem is that a phenomenon, such as that of grading a test, is hard enough for any teacher, experienced or not, and that the valuable opportunity of a discussion in connection with a “real” grading situation could have been important. There is, in our material, not a complete absence of examples that focus subject-specific issues. Some of the statements from the students mark its difference, and we would like to contribute also with some examples of this type. For instance, activities touched upon is "punctuation, paragraphs and stuff" (Anna). Other examples, in this case related to the mathematics subject, are to be found in a discussion between Juanita and her tutor on how to go "from two-dimensional to three-dimensional” and between Kristina and her tutor on "what should be taught and how in mathematics (Kristina). Yet another example is the planning of cross curricular work: When I arrived, the working party had decided on a theme that I became in power of. Me and the team sat discussing what I should deal with, what to do during the English lessons and so on. I got a lot of input from the other teachers when it comes both to the content and on how to carry it out (Kristina). The following utterance shows a situation where the supervisor catches a complexity in mathematics teaching: In math, it makes sense to use the same numbers so that students may compare different results and ask if there is anyone who thinks differently. (Amina) Also interesting for our study is whether students get advice that might be transferred from one situation to another, i. e. advice of a vertical character. There are instances of such an orientation. Juanita confirms this by referring to an advice given, i. e. that it is "important to find out the pupils' prior understanding”. Vol. 4 2014 (Jytte) At several occasions the students themselves express a lack of tutorial concerning subject- specific issues and consequently level criticism of this kind: At several occasions the students themselves express a lack of tutorial concerning subject- specific issues and consequently level criticism of this kind: It was more as if me myself had to come and ask, we had this, to be sure, ten transition from minus and then they thought of plus instead, and I didn’t have a clue when I got there, so then it was me who had to go in and ask her about that. After all it should have been her who had prepared me in such a way and to think… after all, or I could have made them confused there… and to go in with a completely new strategy, sort of. (Jessica) Such an experience is further supported by Amina who asks for more thorough tutor comments: No advice of substance, but methodically, “if you intend to show different ways to solve a number, then use the same figures so that they will be able to recognize and compare the different results”. During a lesson with outdoor mathematics, I was completely nonplussed by a pupil's questions. I went a bit quiet and thought a lot, and then I asked my supervisor if she could help me. "I would have said, quite simply: "Yes, that is one way of thinking, but is there anyone else who might think differently? “" I do not get any advice unless I ask in a very concrete way (Amina). way 8 NordYrk - ISSN 2242-458X – Article Vol. 4 2014 Matilda received a suggestion of a general methodical nature that may still be understood in a decontextualized context: While one group recorded their radio plays, the second group sat drawing sketches that had to do with the radio theatre and then I wanted to get their drawings into a context. Then I got advice on how to tie the tasks together. (Matilda). In some way also the following advice applies for a focus on contents that is transferrable: I got the feeling that the most important was around the content .. how to handle the children if there was someone who sat disturbing at the rear… Should you wait for someone who had gone to the bathroom? Should you write on the board? Should you use a flipchart? Should you use pens of different colours? It was a little more about the practical all around (Johanna). 9 9 NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Vol. 4 2014 Another advice connects to a general aspect of planning: Nordic Journal of Vocational Education and Training Another advice connects to a general aspect of planning: Vol. 4 2014 Vol. 4 2014 Nordic Journal of Vocational Education and Training Vol. 4 2014 significant of a hasty generalization and an over-simplification, lacking support from research. Such justifications concerning feelings, rather than beliefs, indicates tutoring of a horizontal character. Next, language with traits of the horizontal discourse is marked with vagueness. Johanna expresses that "sometimes you hear, many times, that right now we have to put this aside since the group is under process". What it means that a group is “under process” may be hard to comprehend. In the examples below, the concept of ‘flexibility’ can be considered to be vaguely used: We talked a lot about the teaching profession and so, and talked a lot about this with flexibility. That was something he talked about all the time, that you should be flexible, snatch them there and then (Alva). It also appears that an amount of the feedback the students get has an everyday character, including language that is informal, such as Amelia’s experience of being told that she “did great” (above). Relevant to this is also Matilda’ s experience of a similar comment, i.e. "it was good", just like Sara’s: To be honest, I do not think you get very much advice. It’s as if you almost have to pull the response from the supervisor after one’s had the lessons. You get very little advice. They just say that “it is good”. When one asks what was good the answer is still "it’s good". It makes you wonder if they don’t see what it is about. That’s something I have felt during all the periods when I have been out. (Sara) Another partial aspect, connected to language, is the use of metaphors. As already seen, Alva’s tutor said that "You own, you own the class!" Similar metaphorically expressed advice, such as that one should "see all students" (Matilda) are marked by the presence of metaphorical language visible also from previous research of work-integrated learning (Hegender et. al., 2012, discuss this in relation to work-integrated learning). Such language might without doubt have a function. However a problem immediately apparent is that it may also be elusive and difficult for the students to capture. As such, it risks having an impact which turns the teacher profession into something less specialized. The theme of ‘clarity’ is also mentioned by Sara, however in a slightly different way: One should be clear in one’s teaching. NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article My internship supervisor takes up this with the lesson plans and structure. (Anna) Alfred contributes with yet another example, related to a situation that is specific but has the potential of working as an advice of a more general nature: Before they had done a similar job in social studies, and I just jumped in on this The Glass Bead Journey [a novel] and just went on with the work, but then my supervisor said [as if directed to the pupils] "Think of how you finished the social studies job. What was good in that presentation and that job? What could you have done better? Think about that now when you are doing this task". (Alfred). Our final example exhibits a problem in connection to our criteria for tutorials oriented towards a vertical discourse; it does not embed for all aspects of the forthcoming profession. My supervisor has been very focused on maths. Maths has taken up most of the teaching time. It is in relation to this subject we have had our discussions, we are both interested in maths. My last supervisor does not teach the Swedish subject at all and then it will be natural for us to discuss math. Art is something she sees as a necessary evil. "What shall we do during arts lesson today? Oh, I'll surely come up with something”. It's not as if she looks in the subject plan and considers what pupils ought to get regarding different techniques. In mathematics, however, she is very careful about looking at the subject plan (Kristina). Table 5. The aspect of language Table 5. The aspect of language Table 5. The aspect of language The next aspect has to do with language since tutorial activities with orientations to the vertical discourse imply a certain language. For example we expect to find arguments that are based on evidence and proof. Let us take the following shared experience as our point of departure for reasoning on this: The response from my supervisor was often based on an estimation that they had had fun, the pupils had had fun during the lesson … "yes it seems like they thought it was funny … they perceived it as fun anyway" .. I often got remarks on that. (Johanna) The advice Johanna has received should be questioned from a vertically oriented perspective. The advice given, with its primary focus on the pupils ‘having fun’, may be seen as 10 Vol. 4 2014 It’s a bit difficult because you do not know how clear you ought to be neither how much to challenge students. As can be noticed, Sara points out a contradiction connected to the phenomenon of receiving advice. The advice given, dealing with how to be clear in relation to the pupils lacks precision and is in itself unclear. As evident from the citation from Anna (above) there are also situations where the teacher students themselves question the clarity of advice given. It should be stated, though, that what has been referred to here is a possible illustration of how difficult it might be for a tutor to verbalize certain parts of teaching. There are indeed also instances in our material of language of a vertical character. Such language demonstrates concepts that are 11 NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Vol. 4 2014 Table 7. The aspect of evaluation Nordic Journal of Vocational Education and Training Table 7. The aspect of evaluation Nordic Journal of Vocational Education and Training Vol. 4 2014 Finally we pick up the aspect of context, in this case specified with respect to whether the advice offered the student is context-bound or not. From the interviews conducted it seems as if it is mainly context-bound. Amelia has experienced advice that was communicated spontaneously ‘here and now’: My tutor was very good at that. It came at once. So, right after I had talked to the entire class or gone in and helped a pupil she said immediately; “Oh, you did great!”. She was really good at that, so I got feedback constantly. (Amelia) Another student puts her impression as follows: It was the same here, with my supervisor. She could say “You own the class, you own the class!” To get feedback afterwards, then you don’t find it relevant, but now I take it in a different way, and remember it well. (Alva) Diana contributes with a descriptive example: ... I was at another school before where I did not receive any feedback. Not "this was good, this was bad." Nothing, and then I went to another school in springtime, and then I got a new supervisor at another school, and it was: "But this is good. We will solve it. It looks great with the kids" and it was good. (Diana) The feedback that the students depict is apparently contextualized to specific situations and opportunities. It is, in other words, in this respect marked by a horizontal discourse. As is clear, the students seem to appreciate this context-bound feedback. For example, Alfred says that: Often, there was no time immediately after so it was as we said "offset", and then it's not quite as close on, and when you got it right after, maybe it was on a break, it felt so much closer than if you’d put it off it until the afternoon. It's the same when it goes the other way, to "Do remember that ..." than to "Do you remember what you did yesterday?" And you will not even remember yourself what you did. NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Vol. 4 2014 abstract, however more specialized. This fact is supported by the kind of advice Matilda has received: Nordic Journal of Vocational Education and Training Use water as an exogenous force, and then switch to water as a resource and especially water in short supply. That’s something we discussed in the working part (Matilda). Other example does not include concepts, but still suggest specific advice: Then I got a lot of tips on my teaching and my actions. "When you do this, ask this question, do this (Anders) It is of course a delicate matter how precise to be as a tutor. Still, we argue that it may be useful to discuss how to avoid feedback that is too general and how to achieve conversations characterized by accuracy. Table 6. The aspect of organization of the tutorial situation Table 6. The aspect of organization of the tutorial situation There are interviews to be considered as vertically oriented. Such situations, for instance, place the teacher student’s participation more clearly in a professional context indicating a higher degree of decontextualized communication: We had a coordination meeting when the students had gone home, at two o'clock. So we sat discussing what had been good and what I could have done better. (Krister) Also adding to a horizontal discourse are beliefs relying on a kind of relativism, shown in the following: Also adding to a horizontal discourse are beliefs relying on a kind of relativism, shown in the following: Do they make any signals ... no, I think I am allowed to be shaped a bit like I am, and that it's okay that we are different. That is something my last supervisor put much emphasis on, she cannot mould me to be like she is, so I have been allowed to discover for myself how I want to work, the way I want it ... the last supervisor I had … so it has been very freely and openly. (Hjördis) 12 Discussion of the results from empirical, exemplary material We have previously stated that the primary aim of this article is not to study the empirical material from the interviews. However, we would like to draw some conclusions related to the empirical material that we have used to operationalize the analytical tool, which is the focus of this article. The reason for doing so is that we would wish to contribute an example of possible conclusions to be drawn and how to express these conclusions. As far as this aspect is concerned we can, in light of the material studied, see trends that the practical training of teacher education illustrated in several cases contributes to an education for aspiring professionals with low professional skills. Work-integrated learning is considered a key to students’ professional development (1999/2000:135). Thus, the signs we see of a potentially prevailing horizontal discourse is worrying and points towards the need for some action to be taken with regards to the ambitions of the universities working with the practical contents, such as developing a qualified tutorial education, a point which is also highlighted by Lauvås and Handal (2001), Hegender (2010, 2012) and Wedin, Hultman and Schoultz (2012). Furthermore, indications from our study should be discussed in relation to the impact it has on the quality of teacher education and local school cultures. Eriksson (2009) and Ahlström (2012) suggest that contemporary Swedish teacher education actually makes efforts to link academia with teachers professional' activities. However, Wedin, Hultman and Post (2011) pay attention that the tutoring advice to teacher students still can often be understood as “you learn to swim by being thrown into the water”. Suffice to say, our study gives every reason to believe that these interactions need attention. The implications for future professionals should not be underestimated. It is remarkable that students during their work- integrated learning perceive that they are met with different priorities. The consequences of our example suggest that the practical part (work-integrated learning) asks for being placed within a more developed training of supervisors. Placement cannot be about a "place at a school" but rather a case of a meeting a qualified tutor who is able to challenge and communicate all parts of the profession. For teacher training providers, the resources for work-integrated leaning should be earmarked in such a way that there are also resources for the training of trainers. Nordic Journal of Vocational Education and Training Nordic Journal of Vocational Education and Training Vol. 4 2014 as such enabling them to grow professionally. However, there is also a risk that students who face decontextualized feedback do not get knowledge that is transferrable to new situations. NordYrk - ISSN 2242-458X – Article "Sure, I'll try to remember." (Alfred) A possible reason for the students' appreciation of an ad-hoc feedback, in addition to its potential of being easily remembered, might be that it provides the student teacher with a feeling of being capable, a feeling possibly affecting their performance after graduation, and 13 NordYrk - ISSN 2242-458X – Article Vol. 4 2014 “undertheorized” and they purpose teacher education to challenge this lack of theorization. This study should be understood as providing an analytic tool to support teacher educator and students into a more advanced tutoring situation during work-integrated leaning periods. The problematic relation between theory and practice (Shulman, 1998, Bloomfield, Taylor & Maxwell, 2004; Gustavsson, 2008; Dimenäs, 2010) are even more accentuated when tutors tied to teacher education supervise student teachers during work-integrated learning. From this reasoning we have made the assumption that what professionals in the coaching situation communicate, and thus signal as valuable for future teachers to master, make up choices that will affect what is being communicated and emphasized in the tutorial situation, which will have consequences with regards to their future occupation/profession. These consequences of the work-integrated-learning period could for students be a question of maintenance or possibilities to change and develop their future teaching. Whereas the results seem to significantly show a predominance of horizontally oriented advice, it is tempting to further juggle with the idea of what vertically oriented feedback might look like. It would move outside the immediate world of experience and break loose from the context bound. It would grasp the full teacher mission, and consequently treat both what kinds of pupil identities to foster and how to develop highly valued generic skills as well as subject specific contents. It would make use of information as it could be assumed that tutorial activities also relate to other public information connecting to the teacher profession, such as TV-programs, polemical articles, trade magazines and so forth. Moreover, it would relate to cognitively advanced activities and include accurate and specialized language with concepts that one, both as a student teacher and tutor, may transfer between different situations and occasions and as a consequence make progress, leading to the building of a forward-looking and long-term school culture. In other words it would include those elements that enable teacher students to discuss with others in a way that an academic profession such as teaching demands, and in alignment with the professional culture it belongs to. However, this presupposes a general school culture of such a kind. Granström (2012) emphasizes the fact that teachers' activities are not always characterized by a professionalized approach. It would pave the way for social and professional participation, such as public debates on matters of common pedagogical concern. Discussion of the results from empirical, exemplary material Before moving further, we would like to comment also on dilemmas particularly related to the encounters that emerge between tutor and teacher student. The meetings that occur between tutor and student mean that various situations and dilemmas are discussed. Not only the teaching parts and its priorities for the pupils are in focus; the tutor is faced with the tutorial dilemmas, but also of what to give priority in the tutorial discussions. As we have initially touched upon there are some dilemmas connected to work-integrated learning. Teacher placement period has a strong association with the academic part of the training and the question can be raised whether the pictures student teachers receive from the respective activities are consistent. Halse and Malfroy (2010) describe teacher education as 14 NordYrk - ISSN 2242-458X – Article Vol. 4 2014 the teaching profession is ultimately about enabling pupils to have fun or feel safe, or about maintaining classroom discipline. Only a few students mention guidance oriented to aspects such as the subject-specific content. Nor do instances of an ideological awareness appear, neither implicitly nor explicitly. Discussion of the analytical tool At present, Basil Bernstein’s theoretical frames seem to be attracting a renewed interest. Most familiar for every researcher who is interested in educational issues are probably the concepts of classification and framing. The conceptual pair of vertical and horizontal discourse seems to be less well-known, but, we argue, highly useful when the intention is to capture the nature of pedagogical processes. However, in order to make concepts applicable they must inevitably be specified. This is not the least important in relation to Bernstein’s theory testified as it is difficult and abstract although useful. In this article we have specified an analytical tool and tried it out. Our adaptation is done with particular relevance to work integrated learning, an area that has previously been stated as undertheorized. Some clarifications are needed. Firstly, it is obvious that some aspects interfere with each other. Another clarification is the need for an empirical material that is varied. In this case we only had access to the student teachers’ testimonies but the examples had, without doubt, benefitted from other material, such as interviews with the tutors and observations. However, from the empirical data examples we can understand a close connection to the model. Our conclusion is consequently that the analytical tool could serve as a useful instrument for analyzing tutorial dialogs. We also consider the tool useful for studies of policy documents and other text material connected to work-integrated learning. A strength of the tool, we argue, is the fact that it takes into account that teacher training does not clearly belong to a traditionally academic discipline and by following a tradition of seminar there is the risk that tutors are occupied with their everyday practice and student teachers are coloured by their own experiences from being a pupil. Our expectation is that our approach makes it possible not only to capture the nature of work- integrated learning, but also to point out the possibilities and limitations of the tool. Thus, we welcome repeated analyses as well as elaborations of the tool. Bloomfield, D., Taylor, N. & Maxwell, T (2004). Enhancing the link between university and schools through action research on teaching practicum. Journal of Vocational Education and Training, 56 (3), 335-372. Bourne, J. (2004). Framing talc Towards a 'radical visible pedagogy'. In J. Muller, B. Davies & A. Morais (Eds.), Reading Bernstein, Researching Bernstein. London: Routledge Falmer. Breier, M. (2004) Horizontal discourse in law and labour law, in: Muller, J., Davies, B., Morais, A.(Eds) Reading Bernstein. (London and New York, Routledge Farmer Taylor & Francis Group), p. 222-235. Bernstein, B. (1999). Vertical and Horizontal Discourse: an essay. British Journal of Sociology of Education, 20(2), 266-279. Bernstein, B. (2000). Pedagogy, symbolic control and identity: Theory, research, critique. Rowman & Littlefield Publishers. in relation to academic learning]. Pedagogisk forskning i Sverige 17(1 2), 123 132. Bernstein, B. (1990). Class, codes and control. Vol. 4, The structuring of pedagogic discourse. London: Routledge. Ahlström, K.G. (2012). Kopplingen högskole- och verksamhetsförlagd lärarutbildning [Workintegrated learning in relation to academic learning]. Pedagogisk forskning i Sverige 17(1-2), 123-132. Vol. 4 2014 In sum, it would involve tools which support the student and the tutor to move forward together in a conscious way. It would also pave the way for change. The school is expected to be a place for fostering pupils to future responsible and engaged citizens. For example, Granström stresses that it is crucial for teachers to highlight the school's content and its ideological background. As can be shown, the empirical material indicates a tutorial situation not without its problems. It confirms Dimenäs’ (2010) report of work integrated learning as placed in a discussion of conflicts and dichotomies. First it has to be stated that student teachers experience significant differences in the feedback they receive from their supervisor. The results also indicate that the students’ experiences are predominantly placed in line with the horizontal discourse. Depending on the guidance given, we find an indication that the syllabi and subject plans syllabi are something that can be taken for granted and are not explicitly communicated or significantly governing. What is mentioned is to be considered as context-bound and informal (Ahlström, 2012; Hegender 2010, 2012). Some of the student teachers witness tutorials that may just as well signal that 15 NordYrk - ISSN 2242-458X – Article Ahlström, K.G. (2012). Kopplingen högskole- och verksamhetsförlagd lärarutbildning [Workintegrated learning in relation to academic learning]. Pedagogisk forskning i Sverige 17(1-2), 123-132. Bernstein, B. (1990). Class, codes and control. Vol. 4, The structuring of pedagogic discourse. London: Routledge. Bernstein, B. (1999). Vertical and Horizontal Discourse: an essay. British Journal of Sociology of Education, 20(2), 266-279. Bernstein, B. (2000). Pedagogy, symbolic control and identity: Theory, research, critique. Rowman & Littlefield Publishers. Bloomfield, D., Taylor, N. & Maxwell, T (2004). Enhancing the link between university and schools through action research on teaching practicum. Journal of Vocational Education and Training, 56 (3), 335-372. Bourne, J. (2004). Framing talc Towards a 'radical visible pedagogy'. In J. Muller, B. Davies & A. Morais (Eds.), Reading Bernstein, Researching Bernstein. London: Routledge Falmer. Breier, M. (2004) Horizontal discourse in law and labour law, in: Muller, J., Davies, B., Morais, A.(Eds) Reading Bernstein. (London and New York, Routledge Farmer Taylor & Francis Group), p. 222-235. Nordic Journal of Vocational Education and Training Vol. 4 2014 Dimenäs, J. (2010). Beyond dichotomization: a different way of understanding work integrated learning. Journal of Cooperative Education & Internships, 44(2), 43-49. Eriksson, A. (2009). Om teori och praktik i lärarutbildningen. En etnografisk och diskursanalytisk studie [About theory and practice in teacher education. An ethnographic and discourse analytical study]. (Göteborg studies in educational sciences, 275) Göteborg: Acta Universitatis Gothoburgensis. Eriksson, A. (2009). Om teori och praktik i lärarutbildningen. En etnografisk och diskursanalytisk studie [About theory and practice in teacher education. An ethnographic and discourse analytical study]. (Göteborg studies in educational sciences, 275) Göteborg: Acta Universitatis Gothoburgensis. Granström, K. (2012). Tre aspekter på lärares ledarskap i klassrummet [Three aspects on teachers leadership in classroom]. In G. Berg, F. Sundh & C. Wede (ed.), Läraren som ledare: i och utanför klassrummet [Teacher as leader in and outside classroom]. Lund: Studentlitteratur. Granström, K. (2012). Tre aspekter på lärares ledarskap i klassrummet [Three aspects on teachers leadership in classroom]. In G. Berg, F. Sundh & C. Wede (ed.), Läraren som ledare: i och utanför klassrummet [Teacher as leader in and outside classroom]. Lund: Studentlitteratur. n, P. McDonald, M. (2008). Back to the future: Directions for research in teaching and teacher Grossman, P. McDonald, M. (2008). Back to the future: Directions for research in teaching and teacher d i A Ed l R h J l 45 (1) 184 205 Grossman, P. McDonald, M. (2008). Back to the future: Directions for research in teaching and teach education. American Educational Research Journal, 45 (1), 184-205 ( ) ucation. American Educational Research Journal, 45 (1), 184-205 man, P., Hammerness, K. & McDonald, M. (2009). Redifining teaching, Re-imagining teacher education hers and Teaching: Theory and Practice, 15 (2), 273-290 Gustavsson, S. (2008). Motstånd och mening. Innebörd i blivande lärares seminariesamtal [Resistance and meaning. Meaning in future teacher dialogs] . (Göteborg studies in educational sciences, 266) Göteborg: Acta Gustavsson, S. (2008). Motstånd och mening. Innebörd i blivande lärares seminariesamtal [Resistance and meaning. Meaning in future teacher dialogs] . (Göteborg studies in educational sciences, 266) Göteborg: Acta Universitatis Gothoburgensis. Halse, C. & Malfroy, J. (2010). Retheorizing doctoral supervision as professional work. Studies in Higher Education, 14. Hegender, H. (2010). The Assessment of Student Teachers Academic Professional Knowledge in School-Based Teacher Education. Scandinavian Journal of Educational Research, 54(2), 151-171. Hegender, H. (2010). The Assessment of Student Teachers Academic Professional Knowledge in School Based Teacher Education. Nordic Journal of Vocational Education and Training Scandinavian Journal of Educational Research, 54(2), 151-171. Teacher Education. Scandinavian Journal of Educational Research, 54(2), 151-171. nder, H. (2012). Villkor och praxis: Bedömning av lärarstudenters yrkeskunskaper under Hegender, H. (2012). Villkor och praxis: Bedömning av lärarstudenters yrkeskunskaper under verksamhetsförlagd utbildning [Conditions and practice: Assessment of teachers professional skills in work integrated learning]. Nordic Studies in Education, 30(3), 180-197. verksamhetsförlagd utbildning [Conditions and practice: Assessment of teachers professional skills in work integrated learning]. Nordic Studies in Education, 30(3), 180-197. rksamhetsförlagd utbildning [Conditions and practice: Assessment of teachers professional skills in work tegrated learning]. Nordic Studies in Education, 30(3), 180-197. Hultman, G., Schultz, J. & Post, K. (2011). The interplay student teachers - tutors. The clinical education. Educational Research in Sweden 16 (2) pp. 118-137. Hultman, G., Schultz, J. & Post, K. (2011). The interplay student teachers - tutors. The clinical education. Educational Research in Sweden 16 (2) pp. 118-137. Kvale, S. (1996). Interviews - An Introduction to Qualitative Research Interviewing. Thousand Oaks: Sage. Lauvås, P. & Handal, G. (2001). Handledning och praktisk yrkesteori [Supervision and theory of practice]. Lund: Studentlitteratur. Moore, R. and Muller, J. (2002), 'The Growth of Knowledge and the Discursive Gap '. British Journal of Sociology of Education, 23, 627-637. Norlund, A. (2009). Kritisk sakprosaläsning i gymnasieskolan. Didaktiska perspektiv på läroböcker, lärare och nationella prov [Critical reading of non-fiction in upper secondary school. Didactical perspectives on textbooks, teachers and national tests (Göteborg studies in educational sciences 273) Göteborg: Acta Universitatis Norlund, A. (2009). Kritisk sakprosaläsning i gymnasieskolan. Didaktiska perspektiv på läroböcker, lärare och nationella prov [Critical reading of non-fiction in upper secondary school. Didactical perspectives on textbooks, teachers and national tests. (Göteborg studies in educational sciences, 273) Göteborg: Acta Universitatis Gothoburgensis. nationella prov [Critical reading of non-fiction in upper secondary school. Didactical perspectives on textbooks, teachers and national tests. (Göteborg studies in educational sciences, 273) Göteborg: Acta Universitatis Gothoburgensis. teachers and national tests. (Göteborg studies in educational sciences, 273) Göteborg: Acta Universitatis Gothoburgensis. Norlund, A. (2013). ”Varför tycker du man ska ha dödsstraff, då?” Ett sociologisk-didaktiskt redskap för studier av klassrumsdebatter [”Why death penalty?” A sociological and didactical tool for classroom debates]. Educare, 1, 41-67 Norlund, A, Dimenäs, J., Kolback, K. & Wede, C. (2012). Lärares ledarskap i perspektivet av undervisning och bedömning [Teachers leadership in teaching and assessment]. In Berg, G., Sundh, F. & Wede, C. (ed.). Lärares ledarskap [Teachers leadership]. Lund: Studentlitteratur. References Ahlström, K.G. (2012). Kopplingen högskole- och verksamhetsförlagd lärarutbildning [Workintegrated learning in relation to academic learning]. Pedagogisk forskning i Sverige 17(1-2), 123-132. Bernstein, B. (1990). Class, codes and control. Vol. 4, The structuring of pedagogic discourse. London: 16 NordYrk - ISSN 2242-458X – Article Nordic Journal of Vocational Education and Training Regeringens proposition [Government report] 1999/2000:135. En förnyad lärarutbildning [The new teacher education]. Stockholm: Utbildningsdepartementet. Schulman, L.S. (1998). Theory, practice and the education of professionals. The Elementary School Journal, 98 (5), 911-922. Wedin, A.S., Hultman, G. & Schoultz, J. (2012) Influens och konsistens. Om vad som påverkar handledning nder den verksamhetsförlagda utbildningen. Pedagogisk forskning i Sverige 17(1-2), 80-98. Wedin, A.S., Hultman, G. & Schoultz, J. (2012) Influens och konsistens. Om vad som påverkar handledningen under den verksamhetsförlagda utbildningen. Pedagogisk forskning i Sverige 17(1-2), 80-98. Zhang, L.& Han, Z. (2008) Analysis on the Management of College Theachers ' Tacit knowledge 1 (3), 21-24. hang, L.& Han, Z. (2008) Analysis on the Management of College Theachers ' Tacit knowledge 1 (3), 21- 17
W3209327855.txt	https://vestnik-pp.samgtu.ru/1991-8569/article/download/71823/pdf	en		The phenomenon of laziness as a risk factor of high school students' fascination with gadgets	Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ, Psihologo-pedagogičeskie nauki/Vestnik Samarskogo gosudarstvennogo tehničeskogo universiteta. Seriâ: Psihologo-pedagogičeskie nauki	2,021	cc-by	4,436	ПСИХОЛОГИЯ РАЗВИТИЯ УДК 159.9.07 181 DOI: https://doi.org/10.17673/vsgtu-pps.2021.3.13 ФЕНОМЕН ЛЕНИ КАК РИСКОГЕННЫЙ ФАКТОР УВЛЕЧЕННОСТИ СТАРШЕКЛАССНИКОВ ГАДЖЕТАМИ © © Р.И. Суннатова Психологический институт Российской академии образования, Москва, Российская Федерация Поступила в редакцию 15.06.2021 В окончательном варианте 04.09.2021 Для цитирования: Суннатова Р.И. Феномен лени как рискогенный фактор увлеченности старшеклассников гаджетами // Вестник Самарского Государственного Технического Университета. Серия «Психологопедагогические науки». 2021. Т. 18. № 3. С. 181–191. DOI: https://doi.org/10.17673/vsgtu-pps.2021.3.13 В исследовании, представленном в статье, используется термин «увлеченность компьютерными играми и просмотром социальных сетей» как предиктор компьютерной зависимости, которую неправомерно определять как зависимость, но блокирующее влияние которой на учебную активность и продуктивность жизнедеятельности школьников можно констатировать. Исследование осуществлялось в соответствии с экопсихологической моделью становления субъектности — от субъекта восприятия до субъекта продуктивного развития. Целью было изучение возможных связей увлеченности компьютерными играми и социальными сетями с таким состоянием личности, которое в обыденном сознании определяется как лень школьников. В соответствии с пониманием становления субъектности (субъекта продуктивного развития) в экопсихологической модели ключевым условием выступает активность человека. Мы предположили, что отсутствие активности или пассивность школьников 8–11-х классов, возможно, будет условием для возникновения излишней увлеченности компьютерными играми и просмотром социальных сетей. Были использованы две авторские методики: «Личностные особенности как предикторы ненормативного поведения» и «Лень или нежелание учиться», создание которой связано с необходимостью изучения внутриличностных причин пассивности или отсутствия активности в учебной деятельности или феномена «учебной лени» у обучающихся. Подтверждением гипотез служит то, что получены коэффициенты корреляции Спирмена, показывающие статистически значимые связи увлеченности компьютером и соцсетями со всеми выделенными причинами лени, а именно: снижением учебной мотивации; неуверенностью в себе; установкой, что «не от меня все зависит, потому и нечего стараться»; неважным самочувствием; несформированностью осознанной саморегуляции; неудовлетворенностью отношением учителей. Коэффициенты Спирмена — от 0,326 до 0,421 при р < 0,001. В целом полученные результаты подтверждают теоретическую обоснованность разрабатываемых методик и показывают правомерность их использования для психолого-педагогического сопровождения развития и становления субъектности старшеклассников. Ключевые слова: увлеченность гаджетами и социальными сетями, старшеклассники, риски, активность, феномен лени. Благодарности: статья подготовлена в рамках проекта № 19-29-14067 мк «Коммуникативные взаимодействия и субъектность учащихся в условиях цифровизации образования: от экопсихологии к психодидактике». Вестник Самарского Государственного Технического Университета Серия «Психолого-педагогические науки» Том 18 № 3 2021 182 DEVELOPMENTAL PSYCHOLOGY UDC 159.9.07 THE PHENOMENON OF LAZINESS AS A RISK FACTOR OF HIGH SCHOOL STUDENTS' FASCINATION WITH GADGETS © © R.I. Sunnatova Psychological Institute Russian Academy of Education, Moscow, Russian Federation Original article submitted 15.06.2021 Revision submitted 04.09.2021 For citation: Sunnatova R.I. The phenomenon of laziness as a risk factor of high school students' fascination with gadgets. Vestnik of Samara State Technical University. Series Psychological and Pedagogical Sciences. 2021;18(3):181-191. DOI: https://doi.org/10.17673/vsgtu-pps.2021.3.13 In the study presented in the paper, the term passion for computer games and viewing social networks is used as a predictor of computer addiction and, which is not legally defined as addiction, but its blocking effect on the educational activity and productivity of schoolchildren can be stated. The study was carried out in accordance with the ecopsychological model of the formation of subjectivity from the subject of perception to the subject of productive development. The aim was to study the possible links between the passion for computer games and social networks, and such a state of personality, which in everyday consciousness is defined as laziness of schoolchildren. In accordance with the understanding of the formation of subjectivity (the subject of productive development) in the ecopsychological model, the key condition is human activity. We suggested that the lack of activity or passivity of students in grades 8-11 may be a condition for the emergence of excessive enthusiasm for computer games and viewing social networks. Two author’s methods were used: «Personal characteristics as predictors of abnormal behavior» and «Laziness or unwillingness to learn», the creation of which is associated with the need to study the intrapersonal causes of passivity or lack of activity in educational activities or the phenomenon of «learning laziness» in students. The hypotheses are confirmed by the fact that Spearman’s correlation coefficients were obtained, showing statistically significant links between computer and social media involvement with all the identified causes of laziness, namely: reduced learning motivation, lack of self-confidence, attitudes that» everything does not depend on me, so there is nothing to try», poor health, lack of conscious self-regulation, dissatisfaction with the attitude of teachers. Spearman coefficients from .326 to .421 at p <0.001. In general, the results obtained confirm the theoretical validity of the developed methods and show the validity of their use for psychological and pedagogical support for the development and formation of subjectivity of high school students. Keywords: passion for gadgets and social networks, high school students, risks, activity, the phenomenon of laziness. Acknowledgements: the paper was prepared within the framework of the project No. 19-2914067 of the MC «Communicative interactions and subjectivity of students in the conditions of digitalization of education: from ecopsychology to psychodidactics». Введение Использование современных компьютерных технологий практически во всех сферах деятельности человека — уже свершившийся факт. Вместе с несомненным положительным значением компьютеризации и цифровизации образования следует выявить возможные зоны рисков для личностного развития обучающихся, в частности возникновение увлеченности компьютерными играми и просмотром социальных сетей. Зависимость от Интернета — одна из Vestnik of Samara State Technical University Series «Psychological and pedagogical sciences» Vol. 18 No. 3 2021 ПСИХОЛОГИЯ РАЗВИТИЯ 183 актуальных проблем современности, и исследований, посвященных изучению различных ее аспектов, множество как в отечественной, так и в зарубежной научной литературе. Научным сообществом предложено несколько терминов для обозначения нарушений, связанных с неконтролируемо частым использованием Интернета: «Интернет-зависимость, чрезмерное использование Интернета, проблемное использование Интернета» [1, с. 62]. В исследовании, представленном в статье, мы используем термин «увлеченность гаджетами» как предиктор зависимости, поскольку с 2018 г. в международной классификации болезней МКБ-10 интернет-зависимость была выделена как самостоятельное заболевание. Нами рассматривается та степень увлеченности гаджетами и социальными сетями, которую неправомерно определять как зависимость, но блокирующее влияние которой на учебную активность и продуктивность жизнедеятельности школьников можно констатировать. Исследование возможных рисков увлеченности гаджетами и социальными сетями школьников 8–11-х классов осуществлялось в соответствии с экопсихологической моделью становления субъектности от субъекта восприятия до субъекта продуктивного развития В.И. Панова [2]. Целью представленного исследования было изучение связей увлеченности гаджетами и социальными сетями с таким состоянием личности, которое в обыденном сознании определяется как лень, или ленивость школьников. В соответствии с пониманием процесса становления субъектности (субъекта продуктивного развития) в экопсихологической модели ключевым условием выступает активность человека [2]. Мы предположили, что отсутствие активности или пассивность школьника в таком случае, возможно, будет условием для возникновения излишней увлеченности компьютерными играми и просмотром социальных сетей. 1. Обзор литературы В зависимости от подходов (деятельностного, системного, личностного, акмеологического, антропологического, субъектно-генетического и др.) авторы рассматривают субъектность с разных позиций, предлагают различную структуру субъектности, наполняя ее разным содержанием. Общим в этих различающихся позициях является то, что субъектность представляется в виде свойства или характеристики личности, в основе которой лежит отношение человека к себе как к деятелю, источнику продуктивной активности, отражающее понимание своей способности производить значимые преобразования в мире, в других людях, в самом себе. В поиске движущих сил онтогенетического развития В.Э. Чудновский [3] исследовал источники внутренней активности субъекта и ввел понятие «ядро субъективной активности», которое формируется в процессе взаимодействия «внешнего» и «внутреннего»: «…Человеческий индивид в процессе своей жизнедеятельности приобретает такие свойства, которые не предопределены однозначно ни внешними воздействиями, ни внутренними природными данными. Они — результат их взаимодействия, деятельности индивида как единого саморегулирующего целого» [3]. В качестве этого целого выступает субъективная реальность, обладающая относительной самостоятельностью. Она формируется под влиянием объективных условий, но позднее сама начинает влиять на них и создавать себе «малую среду развития», которая свидетельствует об «активности субъективного начала». Вестник Самарского Государственного Технического Университета Серия «Психолого-педагогические науки» Том 18 № 3 2021 184 DEVELOPMENTAL PSYCHOLOGY В работах В.В. Знакова [4], Е.А. Сергиенко [5], направленных на исследование психологии субъекта, не просто констатируется его активный характер, но и обращается внимание на «самосозидательную, самопорождающую, самотрансформирующую природу человеческой субъектности» [4]. «Личность задает направление психического развития, а субъект — его конкретную реализацию через координацию выбора целей и ресурсов индивидуальности человека. Субъектность человека формируется и проявляется в процессе осуществления трех функций: когнитивной, регулятивной и коммуникативной» [5]. Раскрывая суть экопсихологического подхода к изучению становления субъектности, В.И. Панов отмечает, что «процесс обретения психической активности не исчерпывается обретением ею действительного существования в форме психической реальности действия, то есть стадиями становления субъектности. Следующая стадия должна заключаться в том, чтобы освоенное действие превратилось из объекта усвоения (присвоения) в средство дальнейшего проявления психической активности, когда это действие становится субъективным средством выполнения (или освоения) другого действия, а в предельном варианте – творческого самовыражения индивида» [2, с. 116]. Эмпирическая реализация данной модели в исследованиях И.В. Плаксиной [6], М.В. Селезневой [7], А.В. Капцова [8], V.I. Panov, A.V. Kaptsov, E.I. Kolesnikova [9] позволила выявить различные особенности субъектности, характерные для разных стадий ее становления. А.В. Капцовым, Е.И. Колесниковой, М.В. Селезневой был продемонстрирован универсальный (топологический) характер этой модели становления субъектности на разных контингентах обучающихся применительно к ситуациям освоения действия-образца во взаимодействиях с преподавателем [7–9]. Однако при этом осталась не вполне изученной роль личностного ресурса как внутреннего условия развития субъектности обучающихся. Наше исследование нацелено на выявление и изучение особенностей феномена лени у обучающихся как блока активности школьников, способствующего становлению предикторов такого ненормативного поведения, как интернетзависимость. 2. Материалы и методы Эмпирическая часть исследования проводилась в обычной общеобразовательной московской школе. В нем приняли участие обучающиеся 8–11-х классов, всего 296 школьников. Были использованы две авторские методики. Методика, рабочее название которой «Личностные особенности как предикторы ненормативного поведения», включает шкалу «Увлеченность компьютерными играми и просмотром социальных сетей». Для определения степени увлеченности компьютерными играми и социальными сетями нами были выделены четыре фактора, которые и легли в основу шкалы. Эмоциональный аспект. Высокий уровень эмоциональной привлекательности компьютерных игр, общения в соцсетях и просмотров видео для респондента выступает в определенной мере средством разрядки психоэмоционального напряжения, которое приводит к ощущению эмоционального подъема, что является притягательным, привязывающим пользователя к гаджетам или компьютеру. Vestnik of Samara State Technical University Series «Psychological and pedagogical sciences» Vol. 18 No. 3 2021 ПСИХОЛОГИЯ РАЗВИТИЯ 185 Поведенческий аспект позволяет выявить наличие в поведении подростка подчиненности социальным сетям, а не необходимости выполнения учебных заданий, а также семейных обязанностей. Физиологический аспект — наличие физиологических симптомов (напряжение в глазах или в спине, онемение в руках и т. п.), свидетельствующих о том, что необходимо серьезное внимание со стороны близких взрослых к излишней увлеченности обучающегося гаджетами. Личностный аспект — выявление возможности компенсации социальной неадаптированности и трудностей реализации удовлетворения потребностей подростка и его желаний; иными словами, своеобразного ухода от трудностей реальной жизни в более яркую и интересную виртуальную реальность, где удается самореализоваться и обрести необходимую для подростков успешность; выявление его желания нравиться самому себе, что приводит к тому, что взаимодействие с компьютером становится ведущей потребностью в жизни подростка. Результаты описательной статистики по обсуждаемой шкале в целом: асимметрия — 0,099 при ошибке 0,122; эксцесс — 0,382 при ошибке 0,243; коэффициент альфа Кронбаха — 0,796. Более подробно информация по обоснованию опросника и апробации представлена в статье [10]. На следующем этапе апробации опросника будет добавлена еще одна шкала, нацеленная на выявление «трудностей подростка в межличностном взаимодействии со сверстниками». Как правило, неудовлетворенность отношениями в этом возрасте может выступать достаточно серьезным внутренним условием для возможных нарушений в успешности социализации, например уход в себя или/и неприятие себя. На важность этого аспекта указывает не только признание подростков, но и многие данные психологических исследований [11, 13, 18, 20]. Конструирование второй методики связано с необходимостью изучения внутриличностных причин пассивности, отсутствия активности в учебной деятельности или феномена «учебной лени» у обучающихся, что позволит не только понять, в чем специфика лени, проявляемой, как правило, в бездействии, но и вскрыть ее психологическую сущность как эмоционально-действенное состояние личности, определяющее характер поведения и, соответственно, особенности становления субъектности. Также основанием необходимости создания этой методики являются регулярные обращения учителей и родителей к психологам школ с «жалобами» на лень обучающихся. Они признают буквально: «не представляю, что делать с этой ленью, но зато играть в компьютерные игры и часами проводить время в телефоне — не лень!». Скорее всего, такое эмоционально-действенное состояние школьников тоже можно рассматривать как вариант/тип субъектности, который, однако, не способствует развитию личности. На основе анализа литературы (И.С. Якиманская, В.В. Воробьева [12], С.Т. Посохова [13], Е.В. Чугуй, С.А. Кащенко [14], Lenggono, Tentama [15], Madsen [16], Price [17]), посвященной изучению различных толкований лени, мы выделяем следующие возможные причины или условия, предопределяющие проявление лени, которые и составили шкалы методики: 1. Мотивационно-побудительная причина (условно «не хочу») — заключается в отсутствии или слабости смысла, интереса или наличия цели для учебной активности/деятельности. Вестник Самарского Государственного Технического Университета Серия «Психолого-педагогические науки» Том 18 № 3 2021 186 DEVELOPMENTAL PSYCHOLOGY 2. Неуверенность в себе («не смогу») — школьник не уверен в самом себе, в своих силах, в том, что он может успешно учиться. 3. Локус контроля («не надо») — школьник, интерпретируя свои школьные достижения и неудачи как следствие воздействия внешних факторов, откладывает или не делает что-либо, поскольку считает, что все зависит не от него, а от других людей или обстоятельств. 4. Деструктивные психофизиологические состояния человека («не получается»), характеризующиеся переутомлением, сниженным настроением, низким уровнем активации и тонуса. 5. Несформированность системы осознанной саморегуляции поведения и деятельности человека («не умею»). Таким образом, лень может быть связана с негативным самоотношением человека, с нарушением процесса осознанной саморегуляции и т. д.; вместе с тем возможна полидетерминированность лени как характеристики поведения человека. Лень внешне проявляется почти одинаково — блокируется активность человека, не выполняется та или иная деятельность, откладывается выполнение деятельности, а внутренне за проявлением одного и того же поведения лежат различные психологические причины. Такой подход позволяет не только понять, в чем специфика лени, проявляемой, как правило, в бездействии, но и вскрыть ее психологическую сущность как эмоционально-действенное состояние личности, определяющее характер поведения и соответственно особенности становления субъектности. Показатели описательной статистики по этой методике представлены в табл. 1. Таблица 1 Показатели дескриптивной статистики методики «Лень, или Причины нежелания учиться» № Шкала Асиммет рия Эксцесс Альфа Кронбаха Колмогоров – Смирнов 1 Снижение учебной мотивации 0,111 – 0,108 0,791 0,077 2 Неуверенность в себе 0,183 – 0,340 0,773 0,079 3 Экстернальный локус контроля 0,140 –476 0,781 0,073 4 Психофизиологическое состояние –0,231 –394 0,796 0,055 5 Несформированность осознанной саморегуляции 0,349 –265 0,783 0,080 6 Неудовлетворенность отношением учителей –0,037 –088 0,785 0,074 7 Инфантилизация характера школьника 0,178 0,082 0,850 0,079 8 Стремление находиться к комфорте, ничего не делая 0,123 –188 0,795 0,060 Примечание: Ошибка асимметрии .158; ошибка эксцесса .316. 3. Результаты исследования Поскольку результаты дескриптивной статистики позволяют перейти к решению исследовательских задач, но с использованием непараметрического Vestnik of Samara State Technical University Series «Psychological and pedagogical sciences» Vol. 18 No. 3 2021 ПСИХОЛОГИЯ РАЗВИТИЯ 187 коэффициента Спирмена, перейдем к анализу полученных данных (табл. 2). Шкала «Увлеченность компьютерными играми и просмотром социальных сетей» включала субшкалы эмоциональных, поведенческих и личностных особенностей, позволяющие выделить эти характеристики и тем самым обеспечивающие возможность всесторонне изучить механизм излишнего/ деструктивного вовлечения подростка в использование цифровых устройств. Однако результаты описательной статистики не показали того уровня надежности, который позволил бы нам провести анализ по каждой из субшкал в отдельности. Это определило необходимость решения методической задачи в дальнейшем. Но поскольку в целом по шкале результаты надежны, то в этой статье мы представим анализ в обобщенном варианте. Таблица 2 Связи между увлеченностью компьютером и соцсетями и различными условиями проявления лени у учащихся 8–10-х классов Причины лени или нежелания учиться Увлеченность компьютером и соцсетями Снижение учебной мотивации — «Не хочу» 0,326* Неуверенность в себе — «Не смогу» 0,407* Экстернальный локус контроля — «Не от меня зависит» 0,421* Психофизиологическое состояние — «Нет сил, неважное самочувствие» 0,357* Несформированность осознанной саморегуляции 0,340* Неудовлетворенность отношением учителей — «И не буду делать» 0,373 * Стремление находиться к комфорте, ничего не делая 0,359* Примечание: * Уровень значимости при р < 0,001. Как видно из табл. 2, увлеченность компьютерными играми и просмотром социальных сетей имеет статистически значимые связи со всеми выделенными причинами лени, а именно: снижение учебной мотивации; неуверенность в себе; представление, что «не от меня все зависит, потому и нечего стараться»; неважное самочувствие; несформированность осознанной саморегуляции и неудовлетворенность отношением учителей, которая приводит к неприятию учебного предмета и нежеланию его изучать — «и не буду делать»; стремление находиться к комфорте, ничего не делая. Остановимся чуть подробнее на каждой из перечисленных позиций. Мы предположили, что возможна значимая связь между неудовлетворенностью подростков отношением педагогов и увлеченностью компьютерными играми и просмотром социальных сетей. Полученные значения корреляций позволяют утверждать, что характер отношения педагогов можно рассматривать и как ресурс, и как некий блок в ближайшем окружении подростка для возникновения увлеченности компьютерными играми и просмотром социальных сетей в той мере, которая мешает активности и продуктивности учебной деятельности школьников. Результаты нашего исследования подтверждают данные, полученные в исследовании Е.Д. Чижова и К.И. Алексеева [18], в котором показано, Вестник Самарского Государственного Технического Университета Серия «Психолого-педагогические науки» Том 18 № 3 2021 188 DEVELOPMENTAL PSYCHOLOGY что проблемные темы подростков связаны со значимыми другими (с педагогами). Такие характеристики взаимоотношений, как непонимание, отсутствие поддержки и пренебрежительное отношение к чувству собственного достоинства подростков, выступают в качестве условия «отдаления» от значимых других. В исследованиях, проведенных Eunbi Chang, Boyoung Kim [19], Kim, Lee, [20], получены аналогичные результаты, на основе которых авторы утверждают о сдерживающем эффекте позитивного общения между взрослыми и подростками на возникновение компьютерной зависимости. В исследовании Eunbi Chang, Boyoung Kim [19], в котором изучалась связь игровой зависимости с низким уровнем самооценки, получены аналогичные результаты. Авторы цитируемого исследования считают, что низкую самооценку можно рассматривать как фактор риска игровой зависимости. В школе, в которой был проведен опрос, были проведены встречи с обучающимися с целью ознакомления с полученными данными и их обсуждения (с соблюдением правил конфиденциальности). Обсуждались не только причины, ведущие к проявлению лени, но и способы справиться с нежеланием учиться. Аналогичные встречи были организованы с классными руководителями и родителями. На них больший упор был сделан на то, каким образом значимые взрослые могут содействовать нивелированию причин, из-за которых школьники «ленятся» учиться. Обсуждение и заключение В настоящее время довольно распространено волнение и озабоченность родительской общественности внедрением цифровизации не только в сферу образования, но и повседневную жизнь школьников; это, с их точки зрения, может привести к нежелательным изменениям, связанным с возникновением компьютерной зависимости. Подводя итог проведенному исследованию, нацеленному как раз на изучение возможных причин возникновения увлеченности компьютерными играми и просмотром социальных сетей, отметим, что характер отношения значимых взрослых можно рассматривать и как ресурс, и как некий блок в ближайшем окружении старшеклассников для возникновения увлеченности компьютерными играми и просмотром социальных сетей в той мере, которая мешает активности и продуктивности учебной деятельности школьников. По-видимому, чтобы нивелировать возникновение увлеченности компьютерными играми и просмотром социальных сетей, необходимы условия, способствующие конструктивному взаимодействию школьников с педагогами и позитивному самочувствию обучающихся. Известно, что конструктивный характер взаимодействия педагог — ученик способствует формированию учебной мотивации, веры школьника в свои возможности хорошо учиться и формированию осознанной саморегуляции. Полученные результаты показывают, что условиями, предопределяющими становление субъектности старших школьников, может выступать личностный ресурс, а барьером — эмоционально-действенное состояние, блокирующее активность субъекта учебной деятельности. Проведенное исследование также позволяет констатировать перспективность использования создаваемых методик как для решения исследовательских Vestnik of Samara State Technical University Series «Psychological and pedagogical sciences» Vol. 18 No. 3 2021 ПСИХОЛОГИЯ РАЗВИТИЯ 189 задач, так и для ряда аспектов психологического сопровождения учебной деятельности школьников. В целом результаты, представленные в статье, подтверждают теоретическую обоснованность разрабатываемых методик и показывают правомерность их использования для психолого-педагогического сопровождения развития и становления субъектности старшеклассников. СПИСОК ЛИТЕРАТУРЫ 1. Панов В.И., Патраков Э.В. Цифровизация информационной среды: риски, представления, взаимодействия: монография. – М.: Психологический институт РАО; Курск: Университетская книга, 2020. – 199 с. 2. Панов В.И. Субъект и субъектность: от феномена к процессуальности // Психология человека как субъекта познания, общения и деятельности / Отв. ред. В.В. 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Construction measurement of academic procrastination of eleventh grade high school students in Sukoharjo. International Journal of Scientific and Technology Research. 2020. No. 9 (01). Pp. 454–459. 16. Madsen T. The Conception of Laziness and the Characterisation of Others as Lazy. Human Arenas. 2018. No. 1 (3). Рр. 288–304. 17. Price Devon. Laziness Does Not Exist. Medium. https://humanparts.medium.com/lazinessdoes-not-exist-3af27e312d01 (accessed May 23, 2020). 18. Chizhov E.D., Alekseyev K.I. Predstavleniye o smerti i suitsidal’nom povedenii v virtual’nykh soobshchestvakh molodezhi [The concept of death and suicidal behavior in virtual youth communities]. Voprosy psikhologii. 2019. No. 1. Pp. 78–89. 19. Eunbi Chang, Boyoung Kim. School and individual factors on game addiction: A multilevel analysis. International Journal of Psychology International Journal of Psychology, 2019. 20. Kim J., Lee H., Kim J. The effect of academic stress on internet-game addiction and the moderating effect of parent-child positive communication: Focused on the male game addiction group among adolescents. Korean Journal of Family and Culture. 2015. No. 27 (3). Рр. 126–152. Информация об авторе Ранно Иззатовна Суннатова, доктор психологических наук, ведущий научный сотрудник лаборатории экопсихологии развития и психодидактики. Психологический институт Российской академии образования, Москва, Российская Федерация. E-mail: sunrano@mail.ru Information about the author Ranno I. Sunnatova, Doc. Psych. Sci., Senior Research Scientist of Developmental Ecopsychology and Psychodidactics Laboratory. Psychological Institute Russian Academy of Education, Moscow, Russian Federation. E-mail: sunrano@mail.ru Вестник Самарского Государственного Технического Университета Серия «Психолого-педагогические науки» Том 18 № 3 2021
https://openalex.org/W3189333029	https://www.pure.ed.ac.uk/ws/files/221750672/jnnp_2021_327098.full.pdf	English	null	Secondary injury and inflammation after intracerebral haemorrhage: a systematic review and meta-analysis of molecular markers in patient brain tissue	Journal of neurology, neurosurgery and psychiatry	2,021	cc-by	7,999	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 Secondary injury and inflammation after intracerebral haemorrhage: a systematic review and meta-analysis of molecular markers in patient brain tissue Citation for published version: Loan, JJ, Kirby, C, Emelianova, K, Dando, OR, Poon, MT, Pimenova, L, Hardingham, GE, Mccoll, BW, Klijn, CJ, Al-shahi Salman, R, Schreuder, FH & Samarasekera, N 2021, 'Secondary injury and inflammation after intracerebral haemorrhage: a systematic review and meta-analysis of molecular markers in patient brain tissue', Journal of Neurology, Neurosurgery & Psychiatry, pp. jnnp-2021-327098. https://doi.org/10.1136/jnnp-2021-327098 Citation for published version: Link: Link to publication record in Edinburgh Research Explorer Document Version: Publisher's PDF, also known as Version of record Published In: Journal of Neurology, Neurosurgery & Psychiatry ► ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx. doi.org/10.1136/jnnp-2021- 327098). ABSTRACT B k d We assessed risk of bias using a modified Newcastle- Ottawa Scale (mNOS; mNOS score 0–9; 9 indicates low bias), extracted aggregate data, and used random effects meta-analysis to pool associations of molecules where more than two independent case–control studies reported the same outcome and Gene Ontology enrichment analysis to identify over-represented biological processes in pooled sets of differentially expressed molecules (International Prospective Register of Systematic Reviews ID: CRD42018110204).i on August 10, 2021 by guest. Prot http://jnnp.bmj.com/ 98 on 6 August 2021. Downloaded from To identify potential therapeutic targets for ICH, we conducted a systematic review and meta-analysis of inflammation in human brain tissue following ICH with three aims: first, to identify all molecular measures of inflammation reported by published studies of ICH in human brain tissue; second, to quantify pooled associations between individual molecules and ICH compared with controls; and third, to identify associations between ICH and groups of molecules with similar functions, compared with controls. 5Neurosurgery, München Klinik, gGmbH, Munich, Germany 6Department of Neurology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, Netherlands Results Of 7501 studies identified, 44 were included: 6 were case series and 38 were case–control studies (median mNOS score 4, IQR 3–5). We extracted data from 21 491 analyses of 20 951 molecules reported by 38 case–control studies. Only one molecule (interleukin- 1β protein) was quantified in three case–control studies (127 ICH cases vs 41 ICH-free controls), which found increased abundance of interleukin-1β protein after ICH (corrected standardised mean difference 1.74, 95% CI 0.28 to 3.21, p=0.036, I2=46%). Processes associated with interleukin-1β signalling were enriched in sets of molecules that were more abundant after ICH. on August 10, 2021 by guest. Protected by copyright. http://jnnp.bmj.com/ om Correspondence to Dr Neshika Samarasekera, Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh EH8 9YL, UK; neshika.samarasekera@ ed.ac.uk ABSTRACT B k d ABSTRACT Background Inflammatory responses to intracerebral haemorrhage (ICH) are potential therapeutic targets. We aimed to quantify molecular markers of inflammation in human brain tissue after ICH compared with controls using meta-analysis. 1Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh, UK 2Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK 3UK Dementia Research Institute at Edinburgh, The University of Edinburgh, Edinburgh, UK 4The Usher Institute, The University of Edinburgh, Edinburgh, UK 5 g y Methods We searched OVID MEDLINE (1946–) and Embase (1974–) in June 2020 for studies that reported any measure of a molecular marker of inflammation in brain tissue from five or more adults after ICH. We assessed risk of bias using a modified Newcastle- Ottawa Scale (mNOS; mNOS score 0–9; 9 indicates low bias), extracted aggregate data, and used random effects meta-analysis to pool associations of molecules where more than two independent case–control studies reported the same outcome and Gene Ontology enrichment analysis to identify over-represented biological processes in pooled sets of differentially expressed molecules (International Prospective Register of Systematic Reviews ID: CRD42018110204). Results Of 7501 studies identified, 44 were included: 6 were case series and 38 were case–control studies (median mNOS score 4, IQR 3–5). We extracted data from 21 491 analyses of 20 951 molecules reported by 38 case–control studies. Only one molecule (interleukin- 1β protein) was quantified in three case–control studies (127 ICH cases vs 41 ICH-free controls), which found increased abundance of interleukin-1β protein after ICH (corrected standardised mean difference 1.74, 95% CI 0.28 to 3.21, p=0.036, I2=46%). Processes associated with interleukin-1β signalling were enriched in sets of molecules that were more abundant after ICH. 1Centre for Clinical Brain Sciences, The University of Edinburgh, Edinburgh, UK 2Centre for Discovery Brain Sciences, The University of Edinburgh, Edinburgh, UK 3UK Dementia Research Institute at Edinburgh, The University of Edinburgh, Edinburgh, UK 4The Usher Institute, The University of Edinburgh, Edinburgh, UK 5Neurosurgery, München Klinik, gGmbH, Munich, Germany 6Department of Neurology, Donders Institute for Brain, Cognition and Behaviour, Radboud University Medical Centre, Nijmegen, Netherlands Methods We searched OVID MEDLINE (1946–) and Embase (1974–) in June 2020 for studies that reported any measure of a molecular marker of inflammation in brain tissue from five or more adults after ICH. Search strategy and selection criteria Conclusion Interleukin-1β abundance is increased after ICH, but analyses of other inflammatory molecules after ICH lack replication. Interleukin-1β pathway modulators may optimise inflammatory responses to ICH and merit testing in clinical trials. gy JJML searched Ovid MEDLINE (1946) and Ovid Embase (1974) on 18 June 2020 using terms to identify studies of molecular measures of inflamma- tion in brain tissue from human patients with ICH (online supplemental table 1). We did not apply any language limits to searches and did not use hand searching or searches of grey literature. We extracted all records yielded by these searches to Covidence, where duplicates were removed.6 JJML and CK independently screened the titles and abstracts to identify reports of potentially eligible studies for full-text review. We included cohort studies, case– control studies and case series which reported at least one summary molecular measure of inflamma- tion in adult (age ≥16 years) human brain tissue gust 10, 2021 by guest. Protected by copyright. © Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ. guest. Protected by copyright. METHODS Design g We performed a systematic review and meta- analysis. Our study protocol was prospectively regis- tered with the International Prospective Register of Systematic Reviews (CRD42018110204). FHS and NS are joint senior authors. Received 12 May 2021 Accepted 24 July 2021 Secondary injury and inflammation after intracerebral haemorrhage: a systematic review and meta-analysis of molecular markers in patient brain tissue James JM Loan ‍ ‍ ,1,2,3 Caoimhe Kirby,1,2,3 Katherine Emelianova,2,3 Owen R Dando,2,3 Michael TC Poon,1,4 Leisan Pimenova,5 Giles E Hardingham,2,3 Barry W McColl,2,3 Catharina JM Klijn ‍ ‍ ,6 Rustam Al-Shahi Salman,1 Floris HBM Schreuder,6 Neshika Samarasekera1 on August 10, 2021 by guest. Protected by copyright. http://jnnp.bmj.com/ st published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from has been informed by studies of animal models.3 In rodent models, modulators of inflammatory responses to ICH can be used to improve functional outcome.3 Although animal models provide useful mechanistic insights, there are significant differ- ences in immune physiology, haemorrhage induc- tion and haemorrhage evolution between humans and rodents with ICH.4 5 To aid in the design and selection of interventions for clinical trials that translate the benefits of interventions tested in experimental ICH into humans, greater knowledge of disease processes in humans after ICH is needed. To identify potential therapeutic targets for ICH, we conducted a systematic review and meta-analysis of inflammation in human brain tissue following ICH with three aims: first, to identify all molecular measures of inflammation reported by published studies of ICH in human brain tissue; second, to quantify pooled associations between individual molecules and ICH compared with controls; and third, to identify associations between ICH and groups of molecules with similar functions, compared with controls. has been informed by studies of animal models.3 In rodent models, modulators of inflammatory responses to ICH can be used to improve functional outcome.3 Although animal models provide useful mechanistic insights, there are significant differ- ences in immune physiology, haemorrhage induc- tion and haemorrhage evolution between humans and rodents with ICH.4 5 To aid in the design and selection of interventions for clinical trials that translate the benefits of interventions tested in experimental ICH into humans, greater knowledge of disease processes in humans after ICH is needed. 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 on August 10, 2021 by guest. Protected http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 o on August 10 http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from Cerebrovascular disease Data extraction and analysis d d d l g p p p y We tested for Gene Ontology (GO) biological process term enrichment using the package topGO V.2.41.0. GO enrichment analysis was performed for subsets of genes that had positive or negative associations with ICH, partitioned according to the number of studies repeating analysis of the same gene symbol showing the same direction of association. Gene symbols derived from studies that were at high risk of bias (mNOS score 0–3) were excluded from this analysis. This analysis used topGO’s ‘weight01’ algorithm, a mixture of the ‘elim’ and ‘weight’ algorithms which aims to increase the specificity of GO terms identified and to reduce bias arising from dependence of neighbouring GO terms on each other. The enrichment score numerator for each GO term is defined as the number of genes found in a meta-analysis set, and the denominator is the total number of genes annotated for each GO term in the background gene set. Statistical signifi- cance of normalised enrichment is determined using Fisher’s exact test. The background gene set was defined as genes detected at a mean expression of more than one fragment per kilobase million mapped reads by RNA sequencing of seven human brain samples obtained at postmortem following the sudden death of patients due to non-neurological disease (online supplemental table 4). These patients were identified retrospectively from the Sudden Death Brain and Tissue Banks that are part of Medical Research Council Edinburgh and Manchester Brain and Tissue Banks. All patients underwent postmortem examination of brain tissue to exclude acute neurological disease as a cause of death. Cases with frozen tissue available with a sample RNA integrity number >5 were selected on the basis of similar age range (median 72 years, IQR 71.0–74.5) and coexisting small vessel diseases to those associated with ICH (mild–moderate non-amyloid arteriosclerosis, n=4; mild spontaneous CAA, n=1; no abnormality, n=3).7 16 17 RNA was extracted and purified from whole brain tissue homogenate using the RNeasy Lipid Tissue Mini kit (Qiagen, 74804). TruSeq stranded messenger RNA (mRNA)-seq libraries were created per sample (Illumina), which were then each sequenced to a depth of 50 million 50 bp paired-end reads (NovaSeq 6000, Illumina). Read alignment with the human genome was then performed as previously described.18 19 Chord diagrams were produced using the R Circlize package V.0.4.11. JJML and CK independently extracted data from included full texts using standardised proformas (online supplemental table 2). Cerebrovascular disease Cerebrovascular disease we searched Laboratory of Human Retrovirology and Immu- noinformatics Database for Annotation, Visualisation and Inte- grated Discovery Gene ID conversion tool V.6.8, the HGNC multisymbol checker, the US National Centre for Biotechnology Information Gene list and AceView for molecules described by included studies.11–14 We excluded non-specific analyses of multimeric nuclear factor kappa B (NF-κB) and human leuko- cyte antigen (HLA) protein and analyses of lipids which could not be linked to a single gene symbol as well as uncontrolled analyses (figure 1). We classified associations between gene symbols identified in each study as positive if the fold change in expression between ICH and control tissue was greater than 1.5, negative if the fold change was less than 2/3 and neutral if it was equal to or between 2/3 and 1.5.15 Where numerical data could not be extracted from studies, we used reported statistical significance to characterise associations as positive, negative or neutral. If multiple control groups were described, results from control brain tissue from the same case as the ICH were used in preference to control tissue derived from unaffected individuals. We selected peak or trough fold changes where multiple time points were described. If duplicate analyses of overlapping popu- lations were described by multiple study reports, we selected the largest population per analysis. after spontaneous ICH in any language. JJML reviewed the bibli- ographies of each included study for potentially eligible study reports and searched for potentially eligible forward citations using Google Scholar (scholar.google.com). Translators reviewed titles and abstracts before being interviewed by JJML and CK, who made decisions regarding eligibility. We excluded studies of ICH due to an underlying macrovascular cause, trauma or hereditary cerebral amyloid angiopathy (CAA); studies reporting mixed aetiologies of haemorrhage where spontaneous ICH could not be distinguished from other causes; studies of fewer than five cases; conference abstracts; and studies which did not report a molecular measure of brain inflammation (figure 1). We contacted the corresponding authors of studies for which it was unclear if relevant data had been yielded and included these if it was forthcoming. At each screening stage, translators were recruited to translate into English any titles, abstracts and full texts published in other languages (acknowledgements and contributions). A third independent reviewer (FHBMS) made the final decision over inclusion where there were conflicts at the title, abstract and full-text screening stages. INTRODUCTION Stroke due to spontaneous intracerebral haemor- rhage (ICH) causes substantial death and disability.1 There are no effective specific medical treatments for ICH.2 Stroke due to spontaneous intracerebral haemor- rhage (ICH) causes substantial death and disability.1 There are no effective specific medical treatments for ICH.2 To cite: Loan JJM, Kirby C, Emelianova K, et al. J Neurol Neurosurg Psychiatry Epub ahead of print: [please include Day Month Year]. doi:10.1136/jnnp-2021- 327098 Brain inflammation is a potential therapeutic target after ICH. Much of our understanding of the inflammatory response in the brain after ICH Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 1 on August 10, 2021 by guest. Protected b http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from on August 10 http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from Data extraction and analysis d d d l Summary measures of expression of molecules were extracted for ICH cases, control brain tissue from ICH cases that was distant from the haematoma and control brain tissue from non-ICH controls. Data derived from any analytical tech- nique using any molecule were extracted with the exception of analyses of amyloid-beta protein, which we did not consider a marker of neuroinflammatory response to ICH as its accumu- lation is known to often precede ICH onset.7 JJML and CK independently assessed the risk of bias of included case–control studies using a modified Newcastle-Ottawa Quality Assessment Scale for case–control studies (Newcastle-Ottawa Scale (mNOS), online supplemental table 3).8 This assessment assigned points for factors indicating low risk of bias, and scores ranged from 0 to 9. We modified the Newcastle-Ottawa Quality Assessment Scale for use in pathological studies of ICH by introducing three additional point scoring criteria: one for use of control tissue distant to ICH from ICH cases, another for efforts to eliminate or control for potential batch effects in tissue processing and a final one for complete reporting of all cases and control data. We used a single item to assess case definition and ascertainment of exposure. Non-response rate was not assessed. We summarised the characteristics and extracted summary neuroinflamma- tory outcome measures from all included studies in a narrative synthesis. on August 10, 2021 by guest. Prot http://jnnp.bmj.com/ 98 on 6 August 2021. Downloaded from on August 10, 2021 by guest. Protected by copyright. http://jnnp.bmj.com/ rom ugust 10, 2021 by guest. Protected by copyright. We undertook a meta-analysis of associations of individual molecules with ICH where more than two studies had analysed the same molecule and reported numerical measures of associ- ation or expression as well as SE for ICH and control tissue. We used the R package ‘Meta’ V.15–1 in RStudio V.1.3.1093 running R Core V.3.6.1 to undertake random-effects meta- analysis of continuous outcome data and produce forest and funnel plots. For random-effects meta-analysis, we calculated variance of true effects, τ2, using the empirical Bayes estimator9 and used the Hartung and Knapp corrected standardised mean difference as the main summary outcome measure.10 We used the I2 statistic to estimate between-studies heterogeneity. est. Protected by copyright. We next attempted to link all molecules analysed by all included studies to Human Gene Organisation Gene Nomen- clature Committee (HGNC)-approved gene symbols. For this, Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. Individual molecule meta-analysis Just one molecule met criteria for meta-analysis. Interleukin-1β (IL-1β) protein was analysed by three independent studies.22–24 These studies analysed IL-1β abundance in brain tissue obtained during surgical treatment of ICH using 3,3′-diaminobenzidine immunohistochemistry (IHC). As a control sample, two studies used surgically resected brain tissue obtained during surgery for non-haemorrhagic, non-malignant disease,22 24 and the other used tissue that was obtained during ICH surgery that had a margin of at least 1 cm from the haematoma border.23 We scored two of these studies at 423 24 points and the other at 522 out of a maximum of 9 points in our mNOS risk of bias assessment. Figure 2 Chord diagram of replicated findings. This demonstrates which studies conducted analyses of the same gene symbols and indicates that many replicated analyses were derived from two studies.20 21 Segments indicate studies reporting analyses of a gene symbol that has been analysed more than once. Chords link segments reporting the same molecule. Chord width is weighted by risk of bias, with larger width indicating lower risk of bias. Figure 2 Chord diagram of replicated findings. This demonstrates which studies conducted analyses of the same gene symbols and indicates that many replicated analyses were derived from two studies.20 21 Segments indicate studies reporting analyses of a gene symbol that has been analysed more than once. Chords link segments reporting the same molecule. Chord width is weighted by risk of bias, with larger width indicating lower risk of bias. We pooled associations described by these studies of 127 patients and 41 controls using random effects meta-analysis, which showed a statistically significant association of increased IL-1β expression after ICH (SMDc 1.74, 95% CI 0.28 to 3.21; p=0.036; I2=46%; figure 3). All studies reported subgroups of patients who underwent surgery at <6 hours and >6 hours after ICH onset. At <6 hours, there was no statistically significant association of IL-1β with ICH (SMDc 2.44, 95% CI −2.76 to 7.64; p=0.18; I2=88%; online supplemental figure 1A), whereas at >6 hours after ICH onset, IL-1β was associated with ICH compared with control tissue (SMDc 2.31, 95% CI 0.15 to 4.47; p=0.044; I2=70%; online supplemental figure 1B). Funnel plot analysis demonstrated no evidence of publi- cation bias (online supplemental figure 2). Data extraction and analysis d d d l doi:10.1136/jnnp-2021-327098 2 Cerebrovascular disease Cerebrovascu Flow diagram of the study selection. Exclusion of ineligible analyses only. Other eligible analyses from the same study remain ogy; ICH, intracerebral haemorrhage. Figure 1 Flow diagram of the study selection. Exclusion of ineligible analyses only. Other eligible analyses from the same study remain included. GO, Gene Ontology; ICH, intracerebral haemorrhage. Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 3 on August 10, 2021 by guest. Protected http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from on August 1 http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from Cerebrovascular disease Cerebrovascular disease Cerebrovascular disease Figure 2 Chord diagram of replicated findings. This demonstrates which studies conducted analyses of the same gene symbols and indicates that many replicated analyses were derived from two studies.20 21 Segments indicate studies reporting analyses of a gene symbol that has been analysed more than once. Chords link segments reporting the same molecule. Chord width is weighted by risk of bias, with larger width indicating lower risk of bias. abundance, and 135 were from the remaining 36 studies. Full data from one microarray study were available from an online repository.20 However, only published data concerning signifi- cantly differentially associated microarray probes were available for the other microarray study, which did not include measures of SE.21 The corresponding author was unable to provide addi- tional data. Eight analyses conducted by five studies could not be assigned a gene symbol. Gene symbols were identified for the remaining 21 483 analyses of protein and RNA by 37 studies. In this dataset, 539 gene symbols (1134 analyses, in 35 studies) were subject to more than one analysis (figure 2). Numerical measures of effect size and/or SE were not available for 598 anal- yses reported by 24 studies and so could not be used for meta- analysis. The final dataset for meta-analysis comprised 20 885 analyses of 20 850 molecules mapped to 20 823 gene symbols and reported by 15 studies (online supplemental table 7). Individual molecule meta-analysis Although they did not meet criteria for meta-analysis, four other included studies reported analysis of IL-1β mRNA,20 21 23 25 and one used western blotting (WB) to measure IL-1β protein.23 All of these analyses found overall IL-1β expression to be increased after ICH compared with control samples. Two studies conducted analysis by time from onset to death. GO enrichment analysis We next sought to identify GO biological processes that were enriched in sets of gene symbols reported by all controlled studies (figure 1 and table 1). GO terms associated with inflammation and suppression of apoptosis were enriched in pooled sets of gene symbols that were found to be increased in one or more studies (figure 4). The most highly enriched were GO:0043066 negative regulation of apoptotic process (enrichment 36/326, p=7×10−19) and GO:0043312 neutrophil degranulation (enrichment 36/575, p=1×10−5). Conversely, terms associated with neuronal activity were enriched in pooled sets of gene symbols that were found to be These studies reported that IL-1β mRNA and protein abundance were not increased less than 12 hours after ICH onset but were increased at later time points up to 72 hours.23 25 Other molecules were analysed by multiple studies but did not meet criteria for meta-analysis (online supplemental table 6). RESULTS Downloaded from Cerebrovascular disease Table 1 Gene symbols analysed after ICH Analyses (n) Gene symbols (n) Increased >1.5-fold mNOS 0–3 mNOS 4–9 ≥1 analysis 2 324* ≥2 analyses 0 23* ≥3 analyses 0 10* ≥4 analyses 0 6 ≥5 analyses 0 2 Reduced >1.5-fold ≥1 analysis 0 290* ≥2 analyses 0 1 Unchanged ≥1 analysis 3 20 785* ≥2 analyses 3 22* ≥3 analyses 0 2 Gene symbols analysed in controlled studies of human brain tissue after ICH according to the number of analyses of each gene symbol and risk of bias. Gene sets of >10 genes per set used for Gene Ontology enrichment analysis. ICH, intracerebral haemorrhage; mNOS, modified Newcastle-Ottawa Scale. urol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Dow abundance. Five of six mRNA analyses were by microarray and found no association with NFKB1, NFKB2, REL, RELA or RELB subunits with ICH compared with control tissue.20 However, hypothesis-driven analysis of NFKB1 mRNA, by semiquantita- tive reverse transcription PCR (RT-PCR),26 or RELA24 27 and multimeric NF-κB complex22 26 protein by IHC and WB found positive associations with ICH compared with control tissue. Tumour necrosis factor (TNF)-α was subject to eight analyses by six studies. One microarray analysis found no association of TNF with ICH, whereas other analyses using RT-PCR,25 26 quantita- tive RT-PCR,23 IHC24 25 28 and WB23 found positive associations of TNF with ICH tissue compared with control tissue. Eleven studies used terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) to identify double-stranded DNA breaks, which are associated with apoptosis, and all of these determined that it was increased after ICH (online supplemental table 6). Gene symbols analysed in controlled studies of human brain tissue after ICH according to the number of analyses of each gene symbol and risk of bias. Gene sets of >10 genes per set used for Gene Ontology enrichment analysis. ICH, intracerebral haemorrhage; mNOS, modified Newcastle-Ottawa Scale. RESULTS Our searches identified 8886 records, of which 7501 were unique. Of these records, 7218 were excluded after a review of titles and abstracts. Of the 283 full texts reviewed, 239 were excluded (figure 1). In total, we identified 44 eligible study reports for inclusion in our narrative synthesis (online supplemental tables 5 and 6). Six uncontrolled case series were included in the narrative review but were excluded from further analysis. The median mNOS score in the remaining 38 case– control studies was 4 (IQR 3–5, online supplemental table 7). These studies reported the outcomes of 21 491 analyses of 20 951 molecules. A total of 21 356 analyses were derived from two studies20 21 which used microarray to measure RNA transcript on August 10, 2021 by guest. Protected by copyright. http://jnnp.bmj.com/ om 4 Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 Figure 3 Forest plot of pooled independent associations of IL-1β protein with ICH. Tissue analysed by immunohistochemistry. Studies of surgically resected perihaematomal tissue compared with healthy-appearing tissue that was resected on approach to the haematoma23 or from controls undergoing surgery for non-haemorrhagic disease.22 24 ICH, intracerebral haemorrhage; IL-1β, interleukin-1β; SMDc, corrected standardised mean difference. Figure 3 Forest plot of pooled independent associations of IL-1β protein with ICH. Tissue analysed by immunohistochemistry. Studies of surgically resected perihaematomal tissue compared with healthy-appearing tissue that was resected on approach to the haematoma23 or from controls undergoing surgery for non-haemorrhagic disease.22 24 ICH, intracerebral haemorrhage; IL-1β, interleukin-1β; SMDc, corrected standardised mean difference. Figure 3 Forest plot of pooled independent associations of IL-1β protein with ICH. Tissue analysed by immunohistochemistry. Studies of surgically resected perihaematomal tissue compared with healthy-appearing tissue that was resected on approach to the haematoma23 or from controls undergoing surgery for non-haemorrhagic disease.22 24 ICH, intracerebral haemorrhage; IL-1β, interleukin-1β; SMDc, corrected standardised mean difference. Figure 3 Forest plot of pooled independent associations of IL-1β protein with ICH. Tissue analysed by immunohistochemistry. Studies of surgically resected perihaematomal tissue compared with healthy-appearing tissue that was resected on approach to the haematoma23 or from controls undergoing surgery for non-haemorrhagic disease.22 24 ICH, intracerebral haemorrhage; IL-1β, interleukin-1β; SMDc, corrected standardised mean difference. Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 4 on August 10, 2021 by guest. Protected http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. GO enrichment analysis Subunits of the NF-κB complex were subject to 10 analyses by five studies, which measured mRNA20 26 and protein22 24 26 27 1 2 3 Increased Decreased 0 20 40 60 0 20 40 60 0 20 40 60 cellular response to lipoteichoic acid cellular response to oxidised low-density lipoprotein particle stimulus positive regulation of cytokine production involved in inflammatory response positive regulation of amyloid-beta formation membrane protein intracellular domain proteolysis B cell homeostasis MyD88-dependent toll-like receptor signaling pathway cellular response to amyloid-beta neurotrophin TRK receptor signaling pathway positive regulation of neuron apoptotic process extrinsic apoptotic signaling pathway in absence of ligand negative regulation of extrinsic apoptotic signaling pathway via death domain receptors positive regulation of interleukin-8 production cellular response to mechanical stimulus positive regulation of NF-kappaB transcription factor activity negative regulation of neuron apoptotic process response to nicotine positive regulation of I-kappaB kinase/NF-kappaB signaling response to axon injury positive regulation of NIK/NF-kappaB signaling positive regulation of type I interferon production interleukin-1-mediated signaling pathway response to hydrogen peroxide response to drug SRP-dependent cotranslational protein targeting to membrane viral transcription nuclear-transcribed mRNA catabolic process, nonsense-mediated decay translational initiation cytokine-mediated signaling pathway neutrophil degranulation maintenance of presynaptic active zone structure regulation of short-term neuronal synaptic plasticity positive regulation of dendritic spine morphogenesis regulation of long-term neuronal synaptic plasticity social behavior neuromuscular process controlling balance positive regulation of excitatory postsynaptic potential glutamate secretion regulation of synaptic vesicle exocytosis synaptic vesicle endocytosis learning neurotransmitter secretion cytoskeleton organization synapse organization chemical synaptic transmission Number of Genes GO Term Direction Increased Decreased Number of analyses Figure 4 Significantly enriched GO biological process terms according to direction of association with ICH. Top 15 most statistically significantly enriched terms in sets of gene symbols that were subject to at least one, two or three analyses by controlled studies included in our review and found to be increased or decreased after ICH. Ranked by number of genes annotated for each go term. GO, Gene Ontology. on August 10, 2021 by guest. Protected by copyr http://jnnp.bmj.com/ st 2021. Downloaded from Figure 4 Significantly enriched GO biological process terms according to direction of association with ICH. Top 15 most statistically significantly enriched terms in sets of gene symbols that were subject to at least one, two or three analyses by controlled studies included in our review and found to be increased or decreased after ICH. CONCLUSION l In conclusion, we have determined that IL-1β is consistently increased beyond 6 hours after ICH. Since this is the only marker of brain inflammation which has been reported by more than two studies of human brain tissue after ICH, it should be prioritised as a potential therapeutic target after ICH. We have identified biological processes, including negative regulation of apoptosis and neutrophil degranulation, that are potentially associated with ICH. These require further study to confirm their roles in the neuroinflammatory response after ICH. Our secondary analysis of GO enrichment implicates biolog- ical processes that may be activated or suppressed after ICH in humans. In large sets of genes that were increased after ICH, there was enrichment of GO terms associated with neutrophil degranulation and negative regulation of apoptosis. This indi- cates that neutrophils accumulate in the human brain after ICH and are supported by studies of rodent models of ICH, where rapid neutrophil accumulation contributes to blood– brain barrier dysfunction and neuronal injury.34 35 Neutrophils obtained from haematomas drained from patients with ICH exhibit induction of proinflammatory genes, including IL-1β.36 Increased peripheral blood neutrophil counts are associated with worse functional outcome at 3 months after ICH.37 Eleven studies found increased TUNEL signal after ICH compared with control tissue, indicating increased apoptosis after ICH. We found enrichment of the term negative regulation of apoptosis in our study. Molecules associated with enrichment of this term in our study have previously been associated with processes that control or mitigate ICH-induced apoptosis in rodent models of ICH, including immune cell migration, proliferation and phago- cytosis of apoptotic cells.38 on August 10, 2021 by guest. Protected by copyright. http://jnnp.bmj.com/ rom Twitter James JM Loan @James_JM_Loan and Rustam Al-Shahi Salman @ BleedingStroke Twitter James JM Loan @James_JM_Loan and Rustam Al-Shahi Salman @ BleedingStroke Acknowledgements Tissue samples were supplied by The Edinburgh Brain and Tissue bank, which is supported by the Medical Research Council, and the Manchester Brain Bank, which is part of the Brains for Dementia Research programme, jointly funded by Alzheimer’s Research UK and Alzheimer’s Society. Dr Jamie Rose, Dr Jamie McQueen and Professor Tara Spiers-Jones provided RNAseq data for two of the cases used to compile our background gene set for Gene Ontology enrichment analysis. RNA sequencing was performed by Edinburgh Genomics, which is funded by the UK NERC, BBSRC and MRC. Cerebrovascular disease Cerebrovascular disease after ICH is a priority to enable the rational targeting of specific cells or molecules for therapeutic intervention. reduced in single studies. The most highly enriched of these were GO:0007268 chemical synaptic transmission (enrichment 57/467, p=9×10−5) and GO:0050808 synapse organisation (enrichment 31/328, p=1×10−4). Gene symbols contributing to enrichment of multiple GO terms are described in online supplemental figures 3 and 4. Full enrichment tables are provided in online supplemental table 8. reduced in single studies. The most highly enriched of these were GO:0007268 chemical synaptic transmission (enrichment 57/467, p=9×10−5) and GO:0050808 synapse organisation (enrichment 31/328, p=1×10−4). Gene symbols contributing to enrichment of multiple GO terms are described in online supplemental figures 3 and 4. Full enrichment tables are provided in online supplemental table 8. Strengths of our study include the use of a comprehensive search strategy, not restricted by language or date of publication, thorough critical appraisal to determine inclusion and indepen- dent data extraction by two reviewers. We identified many anal- yses of human brain tissue, some of which were not considered by previous narrative reviews of responses to ICH in humans.3 We used a stepwise selection process to determine that just three analyses of one marker were reported with sufficient data to permit meta-analysis. These analyses were conducted by three research groups from different institutions. Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 DISCUSSION I hi In this systematic review of 38 studies of inflammation in human brain tissue after ICH, we identified analyses of 20 951 molecules of which only one—IL-1β protein—has been analysed more than two times and reported by studies that quantified its association with ICH. IL-1β protein is significantly increased from 6 hours after ICH onset.22–24 In addition, we found enrichment of GO terms associated with inflammatory processes, such as negative regulation of apoptotic process and neutrophil degranulation in sets of genes that were increased after ICH. g p Our study also has some limitations. Of the 44 included studies, 42 aimed to address a specific hypothesis and selected targets for analysis on that basis. Using prior knowledge to inform selection of target molecules may have driven selective replication and publication of findings that were predicted to show an association with ICH. Moreover, attempts to confirm pre-existing hypotheses may have driven confirmation bias. These may account for the paucity of replicated analyses of gene symbols which were reduced or unchanged after ICH. Just two studies used a hypothesis-free approach, which was RNA microarray.20 21 These two studies contributed many gene symbols to our enrichment analyses, which risk introducing biases and imprecision associated with older RNA analysis tech- nologies that are susceptible to type II error to our findings.40 Our primary finding of increased IL-1β protein >6 hours after ICH is supported by experimental studies which demonstrated that haemorrhage-induced IL-1β expression in the rodent brain was associated worse functional outcome.29 30 Because there are substantial differences between human ICH and experimental ICH models, it is critical that inflammatory responses to ICH identified by preclinical studies are shown to be conserved in humans prior to the commencement of clinical trials of immune modulators.4 5 An interleukin-1 receptor antagonist is the subject of one ongoing phase II trial for the treatment of ICH,31 another which is being prepared (principal investigator: FHBMS), a phase III trial for subarachnoid haemorrhage32 and a phase II trial in ischaemic stroke.33 The consistent association between ICH and IL-1β expression supports the prioritisation of IL-1β pathway modulators for therapeutic development in ICH. on August 10, 2021 by guest. Prot http://jnnp.bmj.com/ 98 on 6 August 2021. Downloaded from GO enrichment analysis Ranked by number of genes annotated for each go term. GO, Gene Ontology. Figure 4 Significantly enriched GO biological process terms according to direction of association with ICH. Top 15 most statistically significantly enriched terms in sets of gene symbols that were subject to at least one, two or three analyses by controlled studies included in our review and found to be increased or decreased after ICH. Ranked by number of genes annotated for each go term. GO, Gene Ontology. Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 5 on August 10, 2021 by guest. Protected http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from Twitter James JM Loan @James_JM_Loan and Rustam Al-Shahi Salman @ BleedingStroke Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. 20 Rosell A, Vilalta A, García-Berrocoso T, et al. Brain perihematoma genomic profile following spontaneous human intracerebral hemorrhage. PLoS One 2011;6:e16750. Data availability statement Analysis scripts will be shared on contact with the corresponding author after publication. Gene sets derived from human RNAseq analyses may be available to reasonable requests with appropriate ethical approval. Data for the meta-analysis dataset were extracted from published reports or provided by their corresponding authors. 20 Rosell A, Vilalta A, García Berrocoso T, et al. Brain perihematoma genomic profile following spontaneous human intracerebral hemorrhage. PLoS One 2011;6:e16750. 21 Carmichael ST, Vespa PM, Saver JL, et al. Genomic profiles of damage and protection in human intracerebral hemorrhage. J Cereb Blood Flow Metab 2008;28:1860–75. 22 Wang Y-X, Yan A, Ma Z-H, et al. Nuclear factor-κB and apoptosis in patients with intracerebral hemorrhage. J Clin Neurosci 2011;18:1392–5. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. 23 Zhang XW, Wu Y, Wang DK, et al. Expression changes of inflammatory cytokines TNF-α, IL-1β and HO-1 in hematoma surrounding brain areas after intracerebral hemorrhage. J Biol Regul Homeost Agents 2019;33:1359–67. on August 10, 2021 by guest. Prote http://jnnp.bmj.com/ 98 on 6 August 2021. Downloaded from g g g 24 Zhang Z, Liu Y, Huang Q, et al. NF-κB activation and cell death after intracerebral hemorrhage in patients. Neurol Sci 2014;35:1097–102.l 24 Zhang Z, Liu Y, Huang Q, et al. NF-κB activation and cell death after intracerebral hemorrhage in patients. Neurol Sci 2014;35:1097–102. 25 Chen L, Guo F, Li X. Real-Time analysis of inflammatory cytokines and regulatory gene expression in tissues surrounding the hematoma after intracerebral hemorrhage. Neural Regeneration Research 2008;3:1197–202. 26 Gang X, Han Q, Zhao X, et al. Dynamic changes in Toll-like receptor 4 in human Perihematoma tissue after intracerebral hemorrhage. ORCID iDs 29 Lu A, Tang Y, Ran R, et al. Brain genomics of intracerebral hemorrhage. J Cereb Blood Flow Metab 2006;26:230–52. James JM Loan http://orcid.org/0000-0002-6451-9448 James JM Loan http://orcid.org/0000-0002-6451-9448 James JM Loan http://orcid.org/0000-0002-6451-9448 Catharina JM Klijn http://orcid org/0000 0002 8495 4578 on August 10, 2021 by guest. Protected by copyright. http://jnnp.bmj.com/ from Catharina JM Klijn http://orcid.org/0000-0002-8495-4578 Catharina JM Klijn http://orcid.org/0000-0002-8495-4578 30 Sozen T, Tsuchiyama R, Hasegawa Y, et al. Role of interleukin-1beta in early brain injury after subarachnoid hemorrhage in mice. Stroke 2009;40:2519–25. 31 Parry-Jones A. BLOC-ICH: interleukin-1 receptor antagonist in intracerebral haemorrhage. ClinicalTrials.gov, 2020. Available: https://clinicaltrials.gov/ct2/show/ NCT03737344 [Accessed 19 Jan 2021]. Provenance and peer review Not commissioned; externally peer reviewed. World Neurosurg 2018;118:e593–600. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. 27 Wu H, Zhang Z, Hu X, et al. Dynamic changes of inflammatory markers in brain after hemorrhagic stroke in humans: a postmortem study. Brain Res 2010;1342:111–7. 28 Liu B, Hu B, Shao S, et al. CD163/Hemoglobin oxygenase-1 pathway regulates inflammation in hematoma surrounding tissues after intracerebral hemorrhage. J Stroke Cerebrovasc Dis 2015;24:2800–9. on August 10 http://jnnp.bmj.com/ J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp-2021-327098 on 6 August 2021. Downloaded from Cerebrovascular disease REFERENCES 1 Krishnamurthi RV, Ikeda T, Feigin VL. Global, regional and Country-Specific burden of ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage: a systematic analysis of the global burden of disease study 2017. Neuroepidemiology 2020;54:171–9. 1 Krishnamurthi RV, Ikeda T, Feigin VL. Global, regional and Country-Specific burden of ischaemic stroke, intracerebral haemorrhage and subarachnoid haemorrhage: a systematic analysis of the global burden of disease study 2017. Neuroepidemiology 2020;54:171–9. 32 ISRCTN74236229: subcutaneous interleukin-1 receptor antagonist (Sc IL-1ra) in stroke study. Available: http://www.isrctn.com/ISRCTN74236229 [Accessed 18 Jan 2021]. 33 Smith CJ, Hulme S, Vail A, et al. SCIL-STROKE (subcutaneous interleukin-1 receptor antagonist in ischemic stroke): a randomized controlled phase 2 trial. Stroke 2018;49:1210–6. 2 Tamborska A, Poon MTC, Al-Shahi Salman R. Characteristics of randomized trials focusing on stroke due to intracerebral hemorrhage: systematic review. Stroke 2018;49:594–600. 34 Sansing LH, Harris TH, Kasner SE, et al. Neutrophil depletion diminishes monocyte infiltration and improves functional outcome after experimental intracerebral hemorrhage. Acta Neurochir Suppl 2011;111:173–8. 3 Xue M, Yong VW. Neuroinflammation in intracerebral haemorrhage: immunotherapies with potential for translation. Lancet Neurol 2020;19:1023–32. hemorrhage. Acta Neurochir Suppl 2011;111:173–8. 4 Mestas J, Hughes CCW. Of mice and not men: differences between mouse and human immunology. J Immunol 2004;172:2731–8. 35 Moxon-Emre I, Schlichter LC. Neutrophil depletion reduces blood-brain barrier breakdown, axon injury, and inflammation after intracerebral hemorrhage. J Neuropathol Exp Neurol 2011;70:218–35.i gy 5 Kirkman MA, Allan SM, Parry-Jones AR. Experimental intracerebral hemorrhage: avoiding pitfalls in translational research. J Cereb Blood Flow Metab 2011;31:2135–51. 36 Askenase MH, Goods BA, Beatty HE, et al. Longitudinal transcriptomics define the stages of myeloid activation in the living human brain after intracerebral hemorrhage. Sci Immunol 2021;6:eabd6279. 6 Veritas Health Innovation. Covidence. Melbourne, Australia: Veritas Health Innovation, 2020. 6 Veritas Health Innovation. Covidence. Melbourne, Australia: Veritas Health Innovation, 2020. 37 Lattanzi S, Cagnetti C, Provinciali L, et al. Neutrophil-To-Lymphocyte ratio predicts the outcome of acute intracerebral hemorrhage. Stroke 2016;47:1654–7. 7 Samarasekera N, Smith C, Al-Shahi Salman R. The association between cerebral amyloid angiopathy and intracerebral haemorrhage: systematic review and meta- analysis. J Neurol Neurosurg Psychiatry 2012;83:275–81. 7 Samarasekera N, Smith C, Al-Shahi Salman R. The association between cerebral amyloid angiopathy and intracerebral haemorrhage: systematic review and meta- analysis. J Neurol Neurosurg Psychiatry 2012;83:275–81. 38 Taylor RA, Sansing LH. Microglial responses after ischemic stroke and intracerebral hemorrhage. Clin Dev Immunol 2013;2013:1–10. 8 Wells GA, Shea B, O’Connell D. Patient consent for publication Not required. Patient consent for publication Not required. Ethics approval Generation of gene expression data from human brain tissue that was used for derivation of the background gene sets used in this study was undertaken with ethics committee approval (LREC 2003/8/27, 16/ES/0084, 10/ MRE00/23 and 09/H0906/52+5). 18 Dobin A, Davis CA, Schlesinger F, et al. Star: ultrafast universal RNA-seq aligner. Bioinformatics 2013;29:15–21. 19 Liao Y, Smyth GK, Shi W. featureCounts: an efficient General purpose program for assigning sequence reads to genomic features. Bioinformatics 2014;30:923–30. Cerebrovascular disease Stroke Association. FHBMS is supported by a senior clinical scientist grant from the Dutch Heart Foundation (2019T060). NS is supported by an NHS Research Scotland fellowship and the Stroke Association. 10 Harrer M, Cuijpers P, Furukawa T. Pooling Effect Sizes. In: Doing Meta-Analysis in R: A Hands-on Guide, 2019. 11 Huang DW, Sherman BT, Stephens R, et al. David gene ID conversion tool. Bioinformation 2008;2:428–30. Role of the funding source The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The corresponding author had full access to all the data in the study and had the final responsibility for the decision to submit for publication. 12 HUGO gene Nomenclature Committee. Multi-symbol checker. Available: https://www. genenames.org/tools/multi-symbol-checker/ [Accessed 6 Jan 2021]. 13 Thierry-Mieg D, Thierry-Mieg J. AceView: a comprehensive cDNA-supported gene and transcripts annotation. Genome Biol 2006;7 Suppl 1:S12–14. Competing interests JJML participates in an academic mentorship programme supervised by an investigator participating in clinical trials of an IL-1 receptor antagonist who had no knowledge of this study. GEH is a cofounder of Astronautx Ltd and holds equity in the company. He has performed consulting services for the Dementia Discovery Fund and Syncona Ltd. CJMK and FHBMS are preparing a separate clinical trial of an IL-1 receptor antagonist. Financial support is described in the declaration of interests. 14 Gene N. Available: https://www.ncbi.nlm.nih.gov/gene/ [Accessed 6 Jan 2021]. 15 Patterson TA, Lobenhofer EK, Fulmer-Smentek SB, et al. Performance comparison of one-color and two-color platforms within the microarray quality control (MAQC) project. Nat Biotechnol 2006;24:1140–50. p j 16 Samarasekera N, Lerpiniere C, Fonville AF, et al. Consent for brain tissue donation after intracerebral haemorrhage: a community-based study. PLoS One 2015;10:e0135043. 17 Rodrigues MA, E Samarasekera N, Lerpiniere C, et al. Association between computed tomographic biomarkers of cerebral small vessel diseases and long-term outcome after spontaneous intracerebral hemorrhage. Ann Neurol 2021;89:266- 279. CONCLUSION l Translation of text into English language was undertaken by the study authors and the following persons: Alessandra Cardinali, Nina Kozar and Dr Kenichiro Sakaguchi. Rosemary Anderson assisted with manuscript curation. Contributors JJML, CK, ORD, CJMK, RASS, FHBMS and NS: study conceptualisation. JJML, CK, KE, ORD, MTCP, LP, GEH, BWM, CJMK, RASS, FHBMS and NS: data analysis and interpretation. JJML: funding acquisition. JJML, CK, KE, ORD, GEH, BWM, CJMK, RASS, FHBMS and NS: methodology. CJMK, RASS, FHBMS and NS: supervision. JJML: writing—original draft preparation. All: writing—review, editing and approval of the manuscript. Contributors JJML, CK, ORD, CJMK, RASS, FHBMS and NS: study conceptualisation. JJML, CK, KE, ORD, MTCP, LP, GEH, BWM, CJMK, RASS, FHBMS and NS: data analysis and interpretation. JJML: funding acquisition. JJML, CK, KE, ORD, GEH, BWM, CJMK, RASS, FHBMS and NS: methodology. CJMK, RASS, FHBMS and NS: supervision. JJML: writing—original draft preparation. All: writing—review, editing and approval of the manuscript. Funding JJML is supported by a Wellcome Trust fellowship (214145/Z/18/Z). CK is supported by the Wellcome Trust Translational Neuroscience PhD Programme (108890/Z/15/Z). BWM and GEH are supported by the UK Dementia Research Institute, which receives its funding from DRI Ltd, funded by the UK Medical Research Council, Alzheimer’s Society and Alzheimer’s Research UK. BWM receives funding from MRC (MR/L003384/1 and MR/R001316/1) and the Leducq Foundation. MTCP is supported by Cancer Research UK Brain Tumour Centre of Excellence Award (C157/A27589). CJMK is supported by a clinical established investigator grant of the Dutch Heart Foundation (grant number 2012 T077) and an Aspasia grant from The Netherlands Organization for Health Research and Development (ZonMw grant number 015.008.048). RA-SS is supported by the Medical Research Council and the Our enrichment analyses include sets of genes derived from the findings of many studies, although individual genes may have been subject to few analyses. These analyses generate hypotheses concerning processes associated with ICH, which require testing and validation in human brain tissue. The expression of multiple elements of biological processes may be simultaneously analysed using high-throughput analytical approaches, the availability of which has increased substantially over the past decade.39 As such, transcriptomic and proteomic analysis of the human brain Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098 6 REFERENCES The Newcastle-Ottawa scale (NOS) for assessing the quality of nonrandomised studies in meta-analyses. The Ottawa Hospital research Institute. Available: http://www.ohri.ca/programs/clinical_epidemiology/oxford.asp [Accessed 18 Sep 2018]. 39 Manzoni C, Kia DA, Vandrovcova J, et al. Genome, transcriptome and proteome: the rise of omics data and their integration in biomedical sciences. Brief Bioinform 2018;19:286–302. 40 Zhao S, Fung-Leung W-P, Bittner A, et al. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS One 2014;9:e78644. 40 Zhao S, Fung-Leung W-P, Bittner A, et al. Comparison of RNA-Seq and microarray in transcriptome profiling of activated T cells. PLoS One 2014;9:e78644. 9 Sidik K, Jonkman JN. A note on the empirical Bayes heterogeneity variance estimator in meta-analysis. Stat Med 2019;38:3804–16. 7 Loan JJM, et al. J Neurol Neurosurg Psychiatry 2021;0:1–7. doi:10.1136/jnnp-2021-327098
https://openalex.org/W2664186398	https://ejournal.uinsatu.ac.id/index.php/taalum/article/download/553/390	Indonesian	null	Pandangan terhadap Anak dalam Ajaran Islam	Ta'Allum : Jurnal Pendidikan Islam/Ta'allum	2,013	cc-by-sa	5,817	1 M. Quraish Shihab, Mukjizat al-Qur’an: Ditinjau Dari Aspek Kebahasaan, Isyarat Ilmiah, dan Pemberitaan Ghaib, (Bandung: Mizan, 2007), hal. 5. Kata Kunci: Anak, keluarga, Islam Kata Kunci: Anak, keluarga, Islam PANDANGAN TERHADAP ANAK DALAM AJARAN ISLAM Moh. Lutfi Nurcahyono Alumnus Pasca Sarjana UIN Sunan Kalijaga Jurusan Hukum Islam Konsentrasi Hukum Keluarga moh.lutfinurcahyono@yahoo.co.id ABSTRACT Children in a family like banana trees, which bear fruit only once. Through its fruit, banana tree not only can grow up with dignity but also can be affected by disease; withered, thin, and then die with no edible fruit. So are children in a family, they could make their family proud, or they could fall into the darkness and obscurity. Based on this disturbing phenomenon, Islam encourages family as much and as early as possible to direct the development of children so as to grow up with dignity in the world and the hereafter. Pendahuluan “Kenali, cintai, dan amalkan kitab sucimu”1 Penulis sengaja memilih sepenggal buah nasehat dari M. Quraish Shihab di atas bukan tanpa alasan dan penguatan, ide ini muncul karena bertepatan membahas tentang anak dalam pandangan Islam yang secara pasti mengarahkan penulis untuk mencoba memberikan pemaparan tentang anak dengan berangkat dari kitab suci al-Qur’an. Penting juga bahwa cerminan sepenggal nasehat tersebut memberikan pemahaman bahwa hal inilah yang menjadi cerminan bagaimana konsepsi anak dalam Islam khususnya tentang upaya orang tua untuk mengarahkan anak- anaknya menuju jalan yang tentunya sesuai dengan doktrin agama yang diyakini kebenarannya. Kita tidak bisa memungkiri bahwa anak adalah suatu ikon tersendiri dalam keluarga lebih-lebih kehadirannya ditunggu-tunggu sejak lama. Terbukti bagaimana pengalihan kasih sayang antara suami-istri menyatu bagaimana memberikan perhatian secara penuh terhadap anak. Anak bisa memberikan motivasi tersendiri baik bagi sang ayah ataupun sang ibu. Namun begitu keberadaan anak juga tidak jarang memberikan mala petaka yang cukup mengecewakan hati. Lebih-lebih adanya anak hanya memberikan tambahan beban materi maupun mental yang berdampak kualitas dan kuantitas hidup menjadi tidak bermutu. Dengan pola pemosisian anak inilah kemudian munculah berbagai tanggapan dari Islam itu sendiri yaitu adakalanya anak sebagai cobaan atau fitnah, anak sebagai pemberi syafa’at pada hari akhir, anak adalah aset atau investasi Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 146 jangka panjang bagi keluarga, atau ada juga pemahaman yang muncul dalam masyarakat bahwa banyak anak banyak rizki. Untuk itulah pada kesempatan kali ini akan mencoba membahas keterkaitan pemahaman-pemahaman tersebut. Pemahaman yang hanya parsial akan memberikan pemahaman yang akan tumpang tindih lebih-lebih tidak ilmiah. p g Penulis juga akan mengulas pendidikan anak karena hal ini sangat memberikan pengaruh ketika dibenturkan keinginan idealnya orang tua yang memposisikan anak sebagai syafa’at baginya. Kemudian juga berlanjut bagaimana idealnya ahklak seorang anak kepada orang tuanya. Mengingat nasehat dari M.Quraish Shihab di atas, maka penulis dalam pembahasannya berangkat dari ayat-ayat al-Qur’an yang secara khusus mengkaji hal-hal tersebut. dengan menggunakan model pendekatan filsafat etik, karena penulis berusaha menemukan nilai-nilai morality yang terdapat dalam ayat-ayat tersebut.. 2 QS. Al-Imran: 19 3 QS. Yunus: 19 4 QS. Al-Baqarah: 143 5 QS. Al-Imran: 104 6 QS. Al-Imran: 110 Islam dan Keluarga Agama yang paling diridhai di sisi Allah hanyalah Islam.2 Statement yang mungkin cukup menggambarkan sikap apologetic sangat jelas ketika orang yang non-Islam membaca statement ini. Namun, sebagai orang muslim yang amantu billahi dan amalus shalih harus senyatanya bahwa dalam kehidupan tidak hanya klaim bahwa agama yang paling diridlai hanyalah Islam itu mampu membuahkan stigma-stigma positif yang berpotensi untuk memberikan penyelamatan bagi orang lain. Inilah yang kemudian mengarahkan bahwa membumikan nilai-nilai Islam dalam kehidupan masyarakat adalah target utama yang harus dijunjung tinggi. gg Manusia sebagai ummatan wᾱhidatan (umat yang satu)3 sudah sepantasnyalah cita-cita keberadaban adalah momen yang amat penting untuk ditegakkan. Amat benarlah Islam memposisikan sebagai ummatan wasathan (umat yang berada di tengah)4 harus mampu memberikan uswah hasanah (suri tauladan yang baik) kepada yang lain. Hal ini bertujuan untuk memberikan statement bahwa umat Islam adalah umat yang mampu mengimbangi segala aspek kehidupan. Umat yang memilih untuk berada di tengah-tengah semua aspek kehidupan pastilah umat yang harus mampu meramu pemosisian diri sebagai media untuk memberikan kenyamanan kepada semua pihak. Misi yang terang sebagai ummatan wasathan adalah mengajak kepada kebaikan, menyuruh dengan cara yang ma’ruf, dan berpartisipasi untuk ikut serta mencegah segala hal yang mengacu kepada kemungkaran.5 Sehingga sangat tepat sekali ketika ada penyebutan bahwa umat Islam adalah khairu ummah (sebaik-baik umat).6 Bertilik dari hal di atas untuk lebih memerankan tugas Islam secara mendasar maka menanamkan dan membumikan nilai-nilai Islam dalam masyarakat haruslah berangkat dari lingkup kecil dari masyarakat itu sendiri yaitu 2 QS. Al-Imran: 19 3 QS. Yunus: 19 4 QS. Al-Baqarah: 143 5 QS. Al-Imran: 104 6 QS. Al-Imran: 110 14 Pandangan terhadap Anak dalam Ajaran Islam – Moh. Lutfi Nurcahyono 147 keluarga. Keluarga yang sehat baik jasmani maupun rahani tentunya akan mengantarkan masyarakat yang cukup ideal. Masyarakat yang dikatakan ideal adalah masyarakat yang mempunyai elemen keluarga (suami-istri dan anak) yang ayem, tentrem, dan sentosa. Pijakan bagi masyarakat baik lingkup kecil ataupun besar (negara) adalah peranan suami-istri yang mencerminkan kekompakkan. Suami-istri yang kompak pasti akan memunculkan keturunan yang tangguh dan akan memberikan manuver-manuver sebagai problem-solving dalam masyarakat itu sendiri. Peran keluarga yang ideal sebagaimana yang dijelaskan di atas tapatlah sangat menjanjikan apabila diukir dalam bingkaian konsepsi dari mahabbah ke sakinah. Secara alami, seseorang tertarik kepada lawan jenisnya mula-mula melalui pertimbangan jasmani. Suasana saling ketertarikan tersebut membuat yang bersangkutan masuk dalam kubangan “jatuh cinta”, baik sepihak (bertepuk sebelah tangan) atau kedua belah pihak (gayung bersambut). 7 QS. Al-A’raf: 156 8 Nurcholish Madjid, Masyarakat Religious, (Membumikan Nilai-Nilai Islam Dalam Kehidupan Masyarakat), cet ke-2, (Jakarta: Paramadina, 2000), hal. 74. Islam dan Keluarga Hal inilah yang dinamakan mahabbah dalam bahasa arab yang merupakan hubungan pria-wanita yang dalam psikologi Freud berhubungan dengan libido, maksudnya masih diliputi dengan hasrat pemenuhan kebutuhan biologis. Tingkat yang selanjutnya adalah mawaddah yang diartikan jika seseorang memilih pasangan lebih mementingkan hal-hal yang abstrak, misalnya segi kepribadian atau nilai-nilai yang sejenisnya. Mawaddah akan berpotensi menjadi lebih kuat karena segi lahiri atau jasmani tidak terlalu banyak menjadi pertimbangan. Kualitas kepribadian adalah lebih penting dan lebih utama baginya dari pada penampakan fisik. Dari tingkatan mawaddah akan mengantarkan pria-wanita kepada jenjang yang lebih tinggi yaitu rahmah. Rahmah adalah jenis cinta ilahi yang berpangkal dari sifat rahman dan rahim-Nya Allah. Dengan begitu cintanya pria-wanita akan dapat mencapai kualitas kecintaan yang tidak terbatas, yang serba meliputi, murni dan sejati, yang sejalan dengan firman Allah, “Rahmah-ku meliputi segala sesuatu.”7 Berangkat dari rahmah itulah rasa saling tertarik antar manusia dari dua jenis yang diikat dengan perjanjian yang berat (mitsᾱqan ghalidlan) dalam pernikahan yang sah dapat menciptakan suasana keluarga sakinah, yaitu kelurga bahagia yang diliputi rasa tenang, tenteram, dan sentosa yang sempurna.8 Kesakinahan tersebut kemudian akan membuahkan duriyyah yang mampu berperan dalam kehidupan bermasyarakat. Hal itu juga akan menjadikan cerminan bahwa nilai-nilai keIslaman telah merasuk dalam kehidupan masyarakat yang kemudian cita-cita masyarakat religius akan tercapai, baik pada waktu itu maupun pada masa yang akan dihadapi karena keluarga itu juga menurunkan keturunan yang tangguh dan yang akan memberikan maneuver-manuver sebagai problem- solving dalam masyarakat religius itu sendiri. Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 148 Pemahaman-Pemahaman Status Anak Sejarah telah menampilkan bagaimana manusia di dunia ini memposisikan anak-anak yang mereka miliki. Sebagaimana yang dilakukan masyarakat arab jahiliyyah dan kebanyakan masyarakat pada umumnya pada zaman dulu, ketika mendengar bahwa anak yang dilahirkan perempuan maka menjadi hitamlah muka mereka karena malu, akan tetapi jika dikabarkan bahwa anak yang lahir laki-laki maka bahagialah mereka. Inilah cerminan bagaimana kondisi masyarakat di dunia ini mempersepsikan anak sesuai dengan pemahaman konsumtif mereka. Anak bukan sebagai lahan inventasi ke depan, generasi penerus dan meneruskan misi kemanusiaan. Banyak orang mewarisi anak dengan air mata tidak dengan mata air, maka jadilah generasi menjadi generasi yang amburadul dan tidak bermutu baik untuk konsumsi dunia maupun akhirat. Tepatlah gagasan bahwa “janganlah kita wariskan air mata untuk generasi kita akan tetapi wariskan mata air untuk masa depan mereka.” Berikut ini penulis akan memaparkan bagaimana konsepsi anak dengan berangkat dari pemahaman yang telah dipaparkan dalam Islam atau pemahaman yang menggejala dalam masyarakat. Hal ini mengarahkan kita supaya benar-benar faham tentang posisi anak dalam kehidupan dunia yang serba fana ini. 9 QS. Al-Anfal: 29 10 QS. Al-Taghabun: 15 Anak adalah Fitnah Islam sebagaimana yang tercermin dalam al-Qur’an memberikan gambaran-gambaran terkait bagaimana posisi anak itu sendiri. Salah satu dari gambaran tersebut adalah bahwa anak itu adalah fitnah atau dalam redaksi tafsir- tafsir diartikan cobaan (ikhbᾱr dan imtihᾱn) sebagaimana tercermin dalam firman Allah:           Artinya : Dan ketahuilah, bahwa hartamu dan anak-anakmu itu hanyalah sebagai cobaan dan Sesungguhnya di sisi Allah-lah pahala yang besar.9          Artinya: Sesungguhnya hartamu dan anak-anakmu hanyalah cobaan (bagimu), dan di sisi Allah-lah pahala yang besar.10           Artinya : Dan ketahuilah, bahwa hartamu dan anak-anakmu itu hanyalah sebagai cobaan dan Sesungguhnya di sisi Allah-lah pahala yang besar.9          Artinya: Sesungguhnya hartamu dan anak-anakmu hanyalah cobaan (bagimu), dan di sisi Allah-lah pahala yang besar.10 Fitnah dalam konteks harta benda dan anak adalah cobaan yang menyusahkan seseorang untuk melakukan atau meninggalakan, menerima atau mengingkari kaitannya dengan i’tiqad, ucapan, perbuatan, atau persoalan- persoalan lain. Dalam masalah harta bisa menjadi cobaan terhadap seseorang yang bisa mengakibatkan sikap-sikap yang bertentangan dengan kemanusiaan tumbuh subur dalam dirinya. Adapun dalam anak sebagai fitnah karena anak itu merupakan buah hati (tsamratu al-fuᾱd) dan belahan hati (afladzu al-akbᾱd), hal ini sebagaimana pendapat Muhammad Abduh yang dikutip oleh Rasyid Ridha dalam tafsir al-manar bahwa “sebagian kegilaan itu diliputkan oleh Allah dalam Pandangan terhadap Anak dalam Ajaran Islam – Moh. Lutfi Nurcahyono 149 hati para ibu dan bapak, Allah mengarahkan keduanya kepada pendermaan harta, sehat, senang, dan lain sebagainya yang sebenarnya Allah mampu untuk mendermakannya sesuai kehendak keduanya.”11 Dalam hadits yang diriwayatkan Abi Sa’id juga diterangkan bahwa anak itu adalah buah hati yang bisa menimbulkan ketakutan, kebakhilan, dan kesediahan.12 اﻟﻮﻟﺪ ﲦﺮة اﻟﻘﻠﺐ وإﻧﻪ ﳎﺒﻨﺔ ﻣﺒﺨﻠﺔ ﳏﺰﻧﺔ اﻟﻮﻟﺪ ﲦﺮة اﻟﻘﻠﺐ وإﻧﻪ ﳎﺒﻨﺔ ﻣﺒﺨﻠﺔ ﳏﺰﻧﺔ اﻟﻮﻟﺪ ﲦﺮة اﻟﻘﻠﺐ وإﻧﻪ ﳎﺒﻨﺔ ﻣﺒﺨﻠﺔ ﳏﺰﻧﺔ Nurchalish Madjid mengutip pendapat A. Yusuf Ali yang menyatakan bahwa Suatu keluarga besar-banyak anak- pernah dianggap suatu sumber kekuasaan dan kekuatan….maka dalam bahasa Inggris, seseorang dengan banyak anak disebut “kantong panahnya penuh (quiver full)”. Sebagaimana anak panah di panah seorang perkasa, begitu pulalah anak-anak usia mudamu. Bahagiakanlah orang yang kantong panahnya penuh dengan mereka: mereka tidak bakal terhina, melainkan mereka akan berbicara dengan pihak musuh di pintu gerbang. Demikian pula halnya dengar harta dan kekayaan: semuanya itu menambah harga diri, kekuasaan dan pengaruh orang. 11 Rasyid Ridha, Tafsir al-Manar, (Beirut: Daru al-Kutub al-‘Ilmiyah, 1999), hal.538 12 ‘Alau al-Din ‘Ali Ibnu Hisam al-Din al-Muttaqiy al-Hindiy al-Burhanu Furiy, Kanzul al- Amal fi Sunani al-Aqwal wa al-Af’al, (Madinah: Muassasah al-Risalah, 1981), hal. 284 13 Nurchalish Madjid, Masyarakat Religious…,hal. 85-86 14 M. Quraish Shihab, Tafsir al-Mishbah; Pesan, Kesan, dan Keserasian al-Qur’an, cet VI, (Jakarta: Lentera Hati, 2006), hal. 426 Anak adalah Perhiasan Keindahan dunia tidak terlepas dengan hal-hal yang mampu memberikan nuansa tenang dan kepuasan bagi manusia ketika mengarungi kehidupannya. Maha Adil bagi Allah yang memberikan hal-hal yang memang kodratnya menjadi kesukaan manusia. Dihiaskanlah untuk kehidupan dunia ini bagi seorang insan dengan rasa kecintaan kepada wanita, kemudian mendambakan anak-anak, semangat untuk mencari harta berupa emas, perak, kuda pilihan, binatang- binatang ternak, dan sawah lading. Serasa kesurgaan dunia ketika semua hal tersebut mampu diperoleh oleh manusia. Akan tetapi Allah lebih menekankan bahwa semua itu hanyalah kesenangan dunia yang tidak kekal, hanya bagi Allahlah segalanya akan kekal.                                                     Artinya: Dijadikan indah pada (pandangan) manusia kecintaan kepada apa-apa yang diingini, Yaitu: wanita-wanita, anak-anak, harta yang banyak dari jenis emas, perak, kuda pilihan, binatang-binatang ternak dan sawah ladang. Itulah kesenangan hidup di dunia, dan di sisi Allah-lah tempat kembali yang baik (surga).15 Artinya: Dijadikan indah pada (pandangan) manusia kecintaan kepada apa-apa yang diingini, Yaitu: wanita-wanita, anak-anak, harta yang banyak dari jenis emas, perak, kuda pilihan, binatang-binatang ternak dan sawah ladang. Itulah kesenangan hidup di dunia, dan di sisi Allah-lah tempat kembali yang baik (surga).15 Dalam ayat tersebut menyimpulkan tiga kata yang harus mendapat perhatian dengan seksama. Pertama Zuyyina, artinya diperhiaskan. Maksudnya, segala barang yang diingini itu ada baiknya dan ada buruknya, tetapi apabila keinginan telah timbul, yang kelihatan hanya eloknya saja dan lupa akan buruk atau susahnya. Kata kedua ialah Hubb, artinya kesukaan atau cinta. Kata ketiga ialah Syahwat, yaitu keinginan-keinginan yang menimbulkan selera menarik nafsu buat memilikinya. Maka disebutlah enam perkara yang disenangi oleh manusia pada ghalibnya.16 Salah satu dari enam kesenangan dunia adalah anak. Anak sebagaimana diposisikan dalam lingkungan keluarga adalah buah dari kecintaan terhadap perempuan. Syawat yang dimiliki seorang laki-laki akan mengarahkan keinginan yang bersifat batin yaitu dengan memiliki anak. Hai orang-orang beriman, janganlah hartamu dan anak-anakmu melalaikan kamu dari mengingat Allah. Barangsiapa yang berbuat demikian Maka mereka Itulah orang-orang yang merugi.17 Sejarah telah mengukir tentang pemposisian seorang anak khususnya laki-laki. Masyarakat arab jahiliah dengan sangat bangga ketika mempunyai keturunan anak laki-laki. 15 QS. Ali-Imran: 16 16 HAMKA, Tafsir al-Azhar, (Jakarta: PT Pustaka Panjimas, 1986), hal. 117-118 17 QS. Al-Munafiqqun: 9 Anak adalah Fitnah Tetapi kekayaan dan keluarga besar itu adalah suatu ujian dan percobaan. Semuanya dapat berbalik menjadi sumber keruntuhan ruhani, jika salah ditangani, atau jika kecintaan kepada semuanya itu menyisihkan kecintaan kepada Tuhan.13 M. Quraish Shihab menjelaskan bahwa anak menjadi cobaan bukan saja ketika orang tua terdorong oleh cinta kepadanya sehingga ia melanggar, tetapi juga dalam kedudukan anak sebagai amanat Allah swt. Allah menguji manusia melalui anaknya, untuk melihat apakah ia memelihara secara aktif, yakni mendidik dan mengembangkan potensi-potensi anak agar menjadi manusia sebagaimana yang dikehendaki Allah, yakni menjadi hamba Allah sekaligus khalifah di dunia. Mengabaikan tugas ini, adalah salah satu bentuk penghianatan terhadap Allah dan amanat yang dititipkannya kepada manusia. Demikian harta benda, bukan saja menjadi ujian ketika harta itu menjadikan manusia manusia melupakan fungsi sosial harta, atau berusaha meraihnya secara batil, tetapi ia juga adalah ujian dari apakah harta tersebut dipelihara dan dikembangkan sehingga hasilnya berlipat ganda melalui usaha yang halal dan baik.14 Anak dalam artian fitnah (ujian atau cobaan) memberikan pemahaman yang menegaskan bahwa orang tua harus berperan sebagaimana tugasnya mendidik anak secara benar dan sesuai dengan tuntunan agama. Hanya dengan pendidikan yang baik maka kesan anak sebagai fitnah akan tergeser dengan sendirinya. Anak akan menjadikan kekuatan yang memberikan pengaruh baik dalam kedudukan maupun kehormatan. Karena sebagaimana yang dijelaskan oleh A. Yusuf Ali bahwa banyak anak akan menjadikan seseorang penuh dengan busur panah yang bisa berpengaruh dalam masyarakat. Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 150 Anak adalah Perhiasan Hal ini sebagai anggapan bahwa mahkota kehormatan keluarga ada di tangan laki-laki, begitu sebaliknya ketika yang lahir anak perempuan maka muka mereka menjadi hitam karena menanggung malu dengan Pandangan terhadap Anak dalam Ajaran Islam – Moh. Lutfi Nurcahyono 151 kelahiran tersebut. anak perempuan hanya akan mendatangkan aib dan merusak citra kehormatan. Islam datang dengan membawa perubahan terhadap pemahaman tersebut, Nabi Muhammad menunjukkan (sebagaimana beliau sangat menyanyangi putrid-putri beliau yaitu: Fatimah az-Zahra’, Zainab, Ummu Kulsum, dan Ruqaiyah) ajaran Islam yang tidak mendiskriminasikan anak perempuan. Namun, anak sebagaiman yang disinggung di atas sebagai fitnah harus benar-benar mendapatkan perlakuan yang spesial. Spesial karena dia adalah belahan hati, special karena sangat menyenangkan, dan special karena tidak jarang apabila salah dalam memposisikan anak akan malah menambah kesengsaraan batin. Kesenangan secara jelas ketika dia masih kecil sangat menghibur bagi orang tuanya. Tidak jarang baik ibu maupun bapak akan menuai ketentraman batin ketika ditengah keluarga ada si mungil yang menyimbulkan kecintaan yang tulus antara suami-istri tersebut. kesenangan juga akan tampak ketika di hari senja orang tua menuai kebahagiaan batin karena rasa hormatnya anak-anaknya, menuai kesuksesan yang membanggakan, dan terlebih sangat terharu bahkan menangis ketika di waktu senja anak-anaknya datang dengan mencium tangan dan mukanya. Menjadilah anak-anak itu rona-rona ketenangan yan sungguh menjadikan orang tua menangis karena bangga. Akan tetapi lain halnya jika anak-anak yang masih kecil itu sangat menghibur mata kemudian tidak mendapat penanganan yang tepat hanya akan menyisakan kepedihan dan penyesalan bagi orang tuanya. Hal ini diakibatkan karena menilai anak hanya pada eloknya saja tidak pada buruk dan susahnya. 19 M. Quraish Shihab, Wawasan al-Qur’an: Tafsir Maudlu’i Atas Pelbagai Permasalahan Umat, cet VIII, (Bandung: Mizan, 1998), hal. 283 20 QS. Ar-Rum: 30 21 Ahmad Ibnu Hambal Ibnu Abdullah al-Syibani, Musnad Ibnu Hanbal, (tt: Muassisah al- Qurtubah, tt), juz 2, hal. 233. 22 QS. Yunus: 90 Anak adalah Fitrah Kehidupan Setiap bayi yang lahir di dunia ini tercipta tanpa ada pengetahuan secuilpun mengenai apa,siapa, kapan, dimana, bagaimana, dan mengapa dia bisa menghirup udara dunia. Perlahan namun pasti bayi itu kemudian mulai menangkap dengan indera tentang apa-apa yang awalnya dia tidak mengetahui. Dari hal inilah penting untuk dipertegas bahwa bayi itu adalah manusia awal yang kontruks pengetahuaannya dibentuk bagaimana inderawinya menangkap fenomena-fenomena di sekitarnya. Kaitannya dengan kajian bertauhid dalam al-Qur’an menyatakan bahwa fitrahnya manusia itu bertauhid kepada Allah, perjanjian yang telah diikrarkan sewaktu sebelum dia lahir di dunia adalah cerminan bahwa semua manusia dilahirkan dalam keadaan Islam. Dan (ingatlah), ketika Tuhanmu mengeluarkan keturunan anak-anak Adam dari sulbi mereka dan Allah mengambil kesaksian terhadap jiwa mereka (seraya berfirman): "Bukankah aku ini Tuhanmu?" mereka menjawab: "Betul (Engkau Tuhan kami), Kami menjadi saksi". (kami lakukan yang demikian itu) agar di hari kiamat kamu tidak mengatakan: "Sesungguhnya Kami (Bani Adam) adalah orang-orang yang lengah terhadap ini (keesaan Tuhan)".18 Ayat ini menegaskan bahwa manusia menyatakan tentang keesaan Tuhan dan bersaksi bahwa tiada tuhan selain Allah. Akan tetapi pada kenyataannya manusia lalai dengan apa yang telah dia ikrarkan. 18 QS. Al-A’raf: 172 Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 152 Fitrah manusia adalah kejadiaannya sejak semula atau bawaan sejak lahir, karena arti dari pada fitrah itu adalah kata yang diambil dari akar kata al-fathr yang berarti belahan, dan dari makna ini lahir makna-makna lain antara lain “penciptaan” atau “kejadian”.19 Dari pengertian inilah bahwa manusia itu sejak awal kejadiaannya, membawa potensi yang lurus, dan dipahami oleh para ulama sebagai tauhid. Maka hadapkanlah wajahmu dengan Lurus kepada agama Allah; (tetaplah atas) fitrah Allah yang telah menciptakan manusia menurut fitrah itu. tidak ada perubahan pada fitrah Allah. (Itulah) agama yang lurus; tetapi kebanyakan manusia tidak mengetahui.20 Potensi keagamaan yang dibawa manusia adalah al-dīn al-qayyīm. Manusia secara fitrah berpotensi untuk meraih apa yang sudah diberikan Allah kepadanya. Namun begitu kebanyakan manusia tidak mengetahui fitrah yang telah dia miliki sejak lahir. Inilah yang menegaskan bahwa adanya berbagai keyakinan yang berbeda-beda di kalangan manusia tidak terlepas dari kelupaan atas fitrahnya. Sungguh Nabi Muhammad menegaskan bahwa: ﻛﻞ ﻣﻮﻟﻮد ﻳﻮﻟﺪ ﻋﻠﻰ اﻟﻔﻄﺮة ﻓﺄﺑﻮاﻩ ﻳﻬﻮداﻧﻪ وﻳﻨﺼﺮاﻧﻪ أو ﳝﺠﺴﺎﻧﻪ ﻛﻤﺎ ﺗﻨﺘﺞ اﻟﺒﻬﻴﻤﺔ ﻬﺑﻴﻤﺔ ﻫﻞ ﲢﺴﻮن ﻓﻴﻬﺎ ﻣﻦ ﺟﺪﻋﺎء Artinya : Setiap bayi itu dilahirkan sesuai dengan fitrahnya, kedua orang tuanyalah yang menjadikan dia yahudi, nasrani, atau majusi. Anak adalah Fitrah Kehidupan Sebagaimana binatang ternak melahirkan yang sejenisnya apakah engkau menyakini pasti yang terlahir onta?21 ﻛﻞ ﻣﻮﻟﻮد ﻳﻮﻟﺪ ﻋﻠﻰ اﻟﻔﻄﺮة ﻓﺄﺑﻮاﻩ ﻳﻬﻮداﻧﻪ وﻳﻨﺼﺮاﻧﻪ أو ﳝﺠﺴﺎﻧﻪ ﻛﻤﺎ ﺗﻨﺘﺞ اﻟﺒﻬﻴﻤﺔ ﻬﺑﻴﻤﺔ ﻫﻞ ﲢﺴﻮن ﻓﻴﻬﺎ ﻣﻦ ﺟﺪﻋﺎء ﻓﻴﻬﺎ ﻣﻦ ﺟﺪﻋﺎء Artinya : Setiap bayi itu dilahirkan sesuai dengan fitrahnya, kedua orang tuanyalah yang menjadikan dia yahudi, nasrani, atau majusi. Sebagaimana binatang ternak melahirkan yang sejenisnya apakah engkau menyakini pasti yang terlahir onta?21 Patut untuk diperhatikan bahwa keberadaan anak dalam lingkup keluarga dari sisi keberagamaan adalah fitrah yang tidak bisa dipaksakan. Inilah yang kemudian memberikan kesan bahwa amanat yang diberikan oleh Allah kepada orang tua adalah menyangkut bagaimana pembentukan orang tua terhadap anak tersebut. dalam artian apakah nantinya anak akan tumbuh sesuai dengan fitrahnya atau melenceng sesuai kehendak orang tuanya. Kejelasan ini akan berdampak pada keberlanjutan anak menjadi khalifah di bumi ini. Al-dīn al-qayyīm adalah fitrah yang sejalan dengan jati diri manusia, kalau tidak pada masa muda, maka menjelang tutup usiapun akan menyadari tentang al- dīn al-qayyīm. Sebagaimana Fir’aun yang durhaka dan merasa dirinya tuhan pun pada akhirnya bertobat dan ingin beragama, tapi sayang nasi sudah menjadi bubur. Dan Kami memungkinkan Bani Israil melintasi laut, lalu mereka diikuti oleh Fir'aun dan bala tentaranya, karena hendak Menganiaya dan menindas (mereka); hingga bila Fir'aun itu telah hampir tenggelam berkatalah dia: "Saya percaya bahwa tidak ada Tuhan melainkan Tuhan yang dipercayai oleh Bani Israil, dan saya Termasuk orang-orang yang berserah diri (kepada Allah)".22 Orang-orang 15 Pandangan terhadap Anak dalam Ajaran Islam – Moh. Lutfi Nurcahyono 153 yang kafir itu seringkali (nanti di akhirat) menginginkan, kiranya mereka dahulu (di dunia) menjadi orang-orang Muslim.23 Bagi manusia fitrah itu tidak hanya potensi keagamaan akan tetapi menyangkut bagaimana fitrah akal dan jasadnya. Hal ini sebagaimana pendapat Syekh Muhammad al-Thahir Ibnu al-‘Asyur yang menyatakan bahwa: ﺑﺄن اﻟﻔﻄﺮة ﻫﻲ اﻟﻨﻈﺎم اﻟﺬي أوﺟﺪﻩ اﷲ ﰲ ﻛﻞ ﳐﻠﻮق ، واﻟﻔﻄﺮة اﻟﱵ ﲣﺺ ﻧﻮع اﻹﻧﺴﺎن ﻫﻲ ﻣﺎ ﺧﻠﻘﻪ اﷲ ﻋﻠﻴﻪ ﺟﺴﺪاً وﻋﻘﻼً Artinya : Sesungguhnya fitrah itu adalah bentuk dan system yang diwujudkan oleh Allah kepada semua makhluk. Fitrah yang khusus kepada manusia adalah apa yang diciptakan oleh Allah pada manusia yang kaitannya dengan jasmani dan akalnya.24 Anak dilahirkan dengan fitrahnya juga tidak terlepas bagaimana fitrah anak menyangkut jasmani dan akalnya. Adapun orang tua hanya sebagai media untuk mengarahkan, membenarkan dan meluruskan fitrah anak yang pada kenyataannya belum mengetahui dengan pasti. 23 QS. Al-Nah: 2 24 Syekh Muhammad al-Thahir Ibnu al-‘Asyur, al-Tahrir wa al-Tanwir, (tt: Daru al-Nasyr, 1997), juz 21, hal. 90. Anak adalah Fitrah Kehidupan Amanat yang diemban oleh orang tua cukup menjadikan penguat bahwa peran serta orang tua untuk anaknya adalah wajib QS. Al Nah: 2 24 Syekh Muhammad al-Thahir Ibnu al-‘Asyur, al-Tahrir wa al-Tanwir, (tt: Daru al-Nasyr, 1997), juz 21, hal. 90. 23 QS. Al-Nah: 2 Artinya : Dan janganlah kamu membunuh anak-anakmu karena takut kemiskinan. kamilah yang akan memberi rezki kepada mereka dan juga kepadamu. Sesungguhnya membunuh mereka adalah suatu dosa yang besar.25 Anak adalah sumber rizki untuk konteks sekarang harus difahami dengan mengarahkan para orang tua untuk berkenyakinan bahwa banyak anak jangan difahami secara kuantitas, melainkan kualitas anak harus didahulukan. Banyak rizki harus difahami dengan memposisikan anak sebagai lahan investasi jangka panjang. Dengan begitu bukan banyak anak banyak rizki, melainkan kualitas yang dimiliki oleh anak sebagai motifasi untuk meraih rizki. Anak adalah Sumber Rizki Ada keyakinan yang lazimnya difahami oleh masyarakat bahwa banyak anak banyak rizki. Hal ini kemudian memberikan arahan kepada suami-istri untuk mempunyai anak tanpa ada keberencanaan. Sangat tepat sekali jikalau anak sebagai sumber rizki bukan dalam maksud bukan kuantitas anak yang dimiliki. Sebenarnya adanya anak dalam keluarga sangat diidam-idamkan, maka dari itu seorang suami akan mendapatkan suplai semangat bekerja yang lebih ketika persembahan hasil keringatnya tidak hanya untuk belahan jiwanya akan tetapi juga untuk buah hati yang sangat diidam-idamkan. Entah apa yang dimaksud banyak anak banyak rizki dalam konteks sekarang. Penulis hanya bisa memprediksi bahwa adagium tersebut muncul untuk merespon penduduk pada masa pra kemerdekaan dan pasca kemerdekaan yang masih sedikit jumlahnya. Munculnya adagium tersebut kemudian diyakini oleh masyarakat secara turun temurun. Namun, dengan berjalannya waktu adanya program KB (keluarga berencana) dari pemerintah dengan bersemangatkan dua anak cukup harus dipahami oleh sebagian masyarakat yang masih menyakini adagium di atas dengan berkaca kepada filosofisnya. Al-Qur’an secara tegas bahwa keberadaan anak itu tidak menjadikan orang lalu berputus asa yang kemudian meniatkan diri untuk membunuh anaknya karena takut kemiskinan.                Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 154 Artinya : Dan janganlah kamu membunuh anak-anakmu karena takut kemiskinan. kamilah yang akan memberi rezki kepada mereka dan juga kepadamu. Sesungguhnya membunuh mereka adalah suatu dosa yang besar.25 Anak adalah Penolong atau Pemberi Syafa’at bagi Orang Tua g y f g g Sebagaimana penjelasan nabi Muhammad bahwa tidak ada yang paling berharga bagi seseorang ketika meninggal dunia kecuali shadaqah jariah, amal shalih, dan anak shalih yang selalu mendoakannya. Betapa sangat terhinanya ketika seseorang meninggal dalam keadaan tidak mempunyai salah satu dari ketiganya. Penekanan inilah yang sangat memberikan sumbangsih anak kepada orang tua ketika sudah meninggal, tidak ada yang berharga melainkan doa sang anak yang bisa menolong keringanan beban pertanyaan yang diajukan oleh para malaikat. Sudah sepantasnya keberadaan anak ketika masih hidup selalu mendoakan orang tua yang telah meninggal sebagai bukti birrul walidain-nya. Hal tersebut sebenarnya juga tidak terlepas kesuksenan orang tua dalam membimbing dan mengarahkan anak-anaknya. Sehingga sudah sepantasnya balasan dari Allah kepada mereka dengan meyadarkan anak-anaknya untuk mendoakannya. Pertolongan Allah kepada para orang tua juga dalam hal ketika anak yang belum baligh meninggal mendahului orang tuanya maka pada saatnya nanti dia akan memberikan syafa’at kepada mereka karena sudah pasti kesucian jiwanya menuntut dia masuk ke dalam surga. Jiwa yang masih suci sebagaimana fitrahnya kemudian menjadi syafaat bagi orang tuanya. Hal ini penting untuk diperhatikan bahwa, kehilangan dambaan hati lebih-lebih pada waktu kecil pasti akan berdampak cukup berat bagi kedua orang tuanya. Namun begitu, ketika kesabaran dan sikap pasrah kepada Allah yang diutamakan maka sebenarnya secara tidak langsung dan dengan sendirinya dia sudah memutuskan perkara yang tepat karena sesungguhnya dari Allahlah semuanya dan kepada Allahlah semuanya akan kembali. Kesadaran bahwa anak itu murni bukan hak milik orang tua, melainkan keberadaannya pasti sesuai dengan kehendak Allah, jikalau Allah menginginkan ada, maka adalah anak tersebut, dan jika Allah menghendaki meniadakan maka terpisahlah anak tersebut dengan orang tuanya. Hal inilah yang kemudia menjadikan Allah menghadiahi mereka dengan adanya syafaat yang diberikan anak yang telah meninggal dikarenakan mereka menghadapinya dengan penuh kesabaran dan penyandaran yang sepenuhnya hanya kepada Allah. 25 QS. Al-Isra’: 31 155 Pandangan terhadap Anak dalam Ajaran Islam – Moh. Lutfi Nurcahyono 26 QS. Luqman: 13 Hubungan Anak dan Orang Tua dalam Islam Tibalah kita pada pembahasan tentang bagaimana perilaku anak kepada kedua orang tua yang telah bersusah payah mendidiknya dengan pengorbanan yang tiada tara. Al-Qur’an secara tegas menyuruh kepada anak untuk melindungi ketauhidannya dan berbakti kepada kedua orang tua. Sebagaimana yang tertera dalam ayat berikut ini:                           Artinya : Dan Tuhanmu telah memerintahkan supaya kamu jangan menyembah selain Dia dan hendaklah kamu berbuat baik pada ibu bapakmu dengan sebaik-baiknya. jika salah seorang di antara keduanya atau Kedua-duanya sampai berumur lanjut dalam pemeliharaanmu, Maka sekali-kali janganlah kamu mengatakan kepada keduanya Perkataan "ah" dan janganlah kamu membentak mereka dan ucapkanlah kepada mereka Perkataan yang mulia.27                           Artinya : Dan Tuhanmu telah memerintahkan supaya kamu jangan menyembah selain Dia dan hendaklah kamu berbuat baik pada ibu bapakmu dengan sebaik-baiknya. jika salah seorang di antara keduanya atau Kedua-duanya sampai berumur lanjut dalam pemeliharaanmu, Maka sekali-kali janganlah kamu mengatakan kepada keduanya Perkataan "ah" dan janganlah kamu membentak mereka dan ucapkanlah kepada mereka Perkataan yang mulia.27 Ayat ini selain menegaskan ketauhidan hanya kepada Allah, juga memberikan arahan kepada manusia supaya bersikap baik kepada kedua orang tua. Kata ihsᾱna sebagai bentuk ibadah kepada Allah dengan berbakti kepada kedua orang tua memberikan dua penekanan, pertama, memberikan nikmat kepada pihak lain, dan kedua perbuatan baik, karena itu kata “ihsan” lebih luas dari sekedar member nikmat atau nafkah. Maknanya bahkan lebih tinggi dan dalam kandungan makna adil, karena adil adalah memperlakukan orang lain sama dengan perlakuan kepada anda, sedangkan ihsan, memperlakukannya lebih baik dari perlakuannya terhadap anda. 27 QS. Al-Isra’: 23 28 M. Quraish Shihab, Tafsir al-Mishbah…, hal. 442 29 Ibid, hal: 443 Pendidikan Anak Pendidikan merupakan media yang penting untuk mengarahkan peran serta orang tua terhadap anak. Pendidikan yang tepat akan menghasilkan perilaku anak sesuai dengan yang diharapkan umunya para orang tua. Pendidikan yang terbaik bagi anak adalah pendidikan yang mampu mentranformasikan nilai-nilai kekinian dalam bingkaian moralitas yang benar. Sehingga orang tua pada era sekarang mendapat tantangan yang cukup menantang terkait peran sertanya dalam usaha menumbuhkan potensi-potensi fitroh anak agar benar-benar menjadi khalifah di bumi. Kurikulum yang diajarkan kepada anak dalam Islam berangkat dari nilai- nilai ketauhidan. Hal ini sebagai modal dan fondasi dasar yang penting untuk menguatkan kecerdasan emosional dan spiritual anak. Kita telah diberikan contoh bagaimana luqman dalam mengarahkan anaknya pertama kalinya menguatkan ketauhidannya                Artinya : Dan (ingatlah) ketika Luqman berkata kepada anaknya, di waktu ia memberi pelajaran kepadanya: "Hai anakku, janganlah kamu mempersekutukan Allah, Sesungguhnya mempersekutukan (Allah) adalah benar-benar kezaliman yang besar".26 Sikap yang seperti ini yang seharusnya ditekankan pada era saat ini. Kita tau bahwa pendidikan tidak ubahnya sebagai lahan untuk mencari peluang pekerjaan dan itu pantas, namun begitu pendidikan seharusnya tidak meninggalkan tujuan utamanya yaitu menghapus ketidaktahuan bukan memintarkan. Berpijak dari hal inilah penekannya anak untuk diperkenalkan ketauhidan selain untuk meluruskan fitrahnya, hal ini juga berperan untuk menghindarkan dia dari sikap dhalim baik li-nafsihi maupun li ghairihi. Sikap dhalim inilah yang menjadikan anak itu laksanan racun dalam keluarga pada akhirnya yang semakin lama menggerogoti kedua orang tuanya. Keidentikan dhalim dengan syirik kemudian menjadikan alasan bahwa pendidikan ketauhidan adalah yang harus diperkenalkan dan ditanamkan dalam jiwa anak-anak. Setelah mempunyai dimensi kekuatan emosional dan spiritual yang tembal pada anak maka untuk proses yang selanjutnya yaitu menganjurkan para orang tua untuk tau diri dan sadar bahwa anak-anak akan menghadapi masa yang berbeda dengan masa orang tuanya maka dari itu pendidikannyapun harus disesuaikan dengan masanya. Hal seperti ini jauh-jauh telah disabdakan nabi Muhammad bahwa didiklah anak-anakmu sesuai dengan zamannya, kerena mereka akan hidup tidak pada zamanmu. Pendidikan kepada anak bisa saja dengan menggunakan nasehat, seperti ucapan seorang ayah kepada anak-anaknya “Kenali, cintai, dan amalkan kitab sucimu”, atau dengan memberikan uswah hasanah, dan yang paling penting yaitu memberikan kepada mereka dengan luqmatul halᾱl (suapan yang halal). 26 QS. Luqman: 13 Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 156 Hubungan Anak dan Orang Tua dalam Islam Adil adalah mengambil semua hak anda dan atau memberi semua hak orang lain, sedang ihsan adalah memberi lebih banyak daripada yang harus anda beri dan mengambil lebih sedikit dari yang seharusnya anda ambil.28 Oleh karena itulah sebagai anak yang harus difahami terkait bagaimana ihsan (berbuat baik) kepada orang tua yang diperintahkan Islam adalah bersikap sopan-santun kepada keduanya dalam ucapan dan perbuatan sesuai dengan adat kebiasaan masyarakat, sehingga mereka merasa senang terhadap kita, serta mencukupi kebutuhan-kebutuhan mereka yang sah dan wajar sesuai kemampuan kita (sebagai anak).29 Hal ini terlebih ketika mereka telah lanjut usia, sebagai anak harus mampu dan wajib berucap kata dengan ucapan yang paling mulia (karīman). Maksudnya apa-apa yang disampaikan kepada mereka bukan saja yang benar dan tepat, bukan saja juga yang sesuai dengan adat kebiasaan yang baik dalam suatu masyarakat, tetapi ia juga harus yang terbaik dan termulia, dan kalaupun seandainya orang tua melakukan suatu “kesalahan” terhadap anak, maka kesalahan tersebut dianggap tidak ada atau dimaafkan (dalam arti dianggap tidak 157 Pandangan terhadap Anak dalam Ajaran Islam – Moh. Lutfi Nurcahyono 157 pernah ada dan terhapus dengan sendirinya) karena tidak ada orang tua yang bermaksud buruk terhadap anaknya. Demikian makna kariman yang dipesankan kepada anak dalam menghadapi orang tuanya. Sebagai anak yang berbakti kepada kedua orang tua adalah di setiap dia menengadah kepada ilahi rabbi tak lupa selalu berdo’a:      Artinya : "Wahai Tuhanku, kasihilah mereka keduanya, sebagaimana mereka berdua telah mendidik aku waktu kecil". Kemudian timbul pertanyaan, bagaimana jika orang tua itu menyuruh atau mengajarkan perbuatan yang bertentangan dengan norma-norma agama? Apakah sebagai anak harus mentaatinya, atau tidak mentaatinya? Jika tidak mentaati bagaimana cara Islam untuk hal itu? Menjadi anak duharkakah anak tersebut? dalam hal tersebut Islam kemudian mengarahkan kepada manusia untuk selalu taat kepada orang tua dalam kondisi apapun, meskipun orang tua sampai pada batas mengajak anak untuk kesyirikan. Ketika hal tersebut terjadi sebagai anak yang sudah mengerti tentang baik-buruk, halal-haram, dan benar-salah maka sudah semestinya anak tidak mengikuti kemauan orang tua. Sikap protes anak kepada orang tua bukan dengan mengklam secara kasar, akan tetapi tetap dengan mengunakan kata-kata yang sopan lagi manguatkan. 30 QS. Luqman: 15 Penutup Islam pada kenyataannya sangat tepat sekali dalam memposisikan anak dalam lingkup keluarga, baik anak sebagai fitnah, perhiasan, fitrah kehidupan, sumber rizki, penolong dan pemberi syafa’at bagi orang tuanya. Semua hal tersebut memberikan asupan-asupan nilai yang perlu dipertimbangkan oleh para orang tua di saat era yang menomorduakan pendidikan keluarga, kesibukan masing-masing suami-istri akan mengurangi rasa perhatiannya kepada si buah hati. Menjadilah buah hati itu tumbuh sesuai dengan lingkungan yang dia hinggapi, sangat beruntung jika lingkungannya baik, akan tetapi sangat menyayangkan jika dia terjebak dalam kubangan modernitas, konsumtif, matrealis, dan hedonis. Hal inilah yang kemudian menempatkan persepsi-persepsi anak dalam Islam menjadi penting untuk mengingatkan kembali para orang tua yang mempunyai kecenderungan acuh tak acuh kepadanya. Hubungan Anak dan Orang Tua dalam Islam Sebagaimana hal ini dijelaskan dalam al-Qur’an: Dan jika keduanya memaksamu untuk mempersekutukan dengan aku sesuatu yang tidak ada pengetahuanmu tentang itu, Maka janganlah kamu mengikuti keduanya, dan pergaulilah keduanya di dunia dengan baik, dan ikutilah jalan orang yang kembali kepada-Ku, kemudian hanya kepada-Kulah kembalimu, Maka Kuberitakan kepadamu apa yang telah kamu kerjakan.30 j Sampai kapanpun bahwa rasa hormat anak kepada orang tua memang tidak bisa diabaikan. Dalam kondisi apapun sikap hormat dan santun tetap menjadi perintah Allah untuk dipraktekkan anak kepada orang tua. Meski demikian rasa ingin mendapatkan rahmat dari Allah adalah yang menjadi muara semua hal itu. Hal inilah yang kemudian menguatkan ketika adanya orang tua kepada anak malah menjauhkan dari penggapaian rahmat Allah maka anak boleh menolak dan tetap berkosisten menjaga akhlakul karimahnya. Karena ketaatan itu hanya dapat dituntut hanya jika orang tua benar-benar yakin bahwa ia berada dalam kebenaran (al-haqq) dan kebaikan (al-ma’rūf), serta jelas tidak dalam kepalsuan (al-bᾱthil) dan kejahatan (al-munkar). Tetapi orang tua tetap berhak atas perilaku baik anak mereka, dalam bentuk tingkah laku dan sikap hormat penuh kasih-sayang. Sehingga dapat ditarik kesimpulan tentang perilaku anak kepada orang tua adalah, pertama, larangan berkata kotor dan tidak pantas kepada orang tua, lebih-lebih ketika salah satu atau keduanya sudah lanjut usia, akan tetapi harus berucap dengan kata-kat yang halus. Kedua, merendahkan diri sebagai bentuk kesopanan dan cinta kasih. Ketiga, selalu berdoa dengan penuh harapan kepada Allah agar merahmati keduanya dengan penuh kasih sayang sebagaimana keduanya telah Ta’allum, Volume 01, Nomor 2, Nopember 2013: 145-158 158 berbuat baik kepada anak di waktu kecil. Hal inilah yang perlu kita renungkan bersama dan resapi yang kemudian kita bisa memperbaiki seberapa jauh sikap hormat kita kepada keduanya. DAFTAR PUSTAKA al-Burhanu Furiy, ‘Alau al-Din ‘Ali Ibnu Hisam al-Din al-Muttaqiy al-Hindiy, Kanzul al-Amal fi Sunani al-Aqwal wa al-Af’al, Madinah: Muassasah al- Risalah, 1981 al-Syibani, Ahmad Ibnu Hambal Ibnu Abdullah, Musnad Ibnu Hanbal, t.tp.: Muassisah al-Qurtubah, tt al-‘Asyur, Syekh Muhammad al-Thahir Ibnu, al-Tahrir wa al-Tanwir, t.tp.: Daru al-Nasyr, 1997 HAMKA, Tafsir al-Azhar, Jakarta: PT Pustaka Panjimas, 1986 Madjid, Nurcholish, Masyarakat Religious (Membumikan Nilai-Nilai Islam Dalam Kehidupan Masyarakat), cet ke-2, Jakarta: Paramadina, 2000 Ridha, Rasyid, Tafsir al-Manar, Beirut: Daru al-Kutub al-‘Ilmiyah, 1999 Shihab, M. Quraish, Mukjizat al-Qur’an: Ditinjau Dari Aspek Kebahasaan, Isyarat Ilmiah, dan Pemberitaan Ghaib, Bandung: Mizan, 2007 --------, Tafsir al-Mishbah; Pesan, Kesan, dan Keserasian al-Qur’an, cet VI, Jakarta: Lentera Hati, 2006 --------, Wawasan al-Qur’an: Tafsir Maudlu’i Atas Pelbagai Permasalahan Umat, cet VIII, Bandung: Mizan, 1998
https://openalex.org/W2998314083	https://link.springer.com/content/pdf/10.1007/s11096-019-00946-9.pdf	English	null	Perspectives of future pharmacists on the potential for development and implementation of pharmacist prescribing in Qatar	International journal of clinical pharmacy	2,020	cc-by	10,382	* Mohammad Issam Diab mohamed.diab@qu.edu.qa Impact on practice and should be considered when developing prescribing frameworks for Qatar. • Barriers to implementing pharmacist prescribing in Qatar must be addressed by various stakeholders to advance pharmacy practice and further optimize the utilization of pharmacy workforce. • Further changes to the pharmacy curriculum and pharma- cist prescribing training program in Qatar are warranted in order to improve pharmacists’ competence in prescrib- ing practice prior to its implementation. • Further changes to the pharmacy curriculum and pharma- cist prescribing training program in Qatar are warranted in order to improve pharmacists’ competence in prescrib- ing practice prior to its implementation. • Prescribing models such as collaborative and supple- mentary prescribing are the most supported models • Prescribing models such as collaborative and supple- mentary prescribing are the most supported models Mohammad Issam Diab1 · Angham Ibrahim1 · Oraib Abdallah2 · Alla El‑Awaisi1 · Monica Zolezzi1 · Rwedah Anwar Ageeb1 · Wishah Hamza Imam Elkhalifa1 · Ahmed Awaisu1 Received: 5 June 2019 / Accepted: 26 November 2019 / Published online: 2 January 2020 © The Author(s) 2019 1 College of Pharmacy, QU Health, Qatar University, P.O. Box 2713, Doha, Qatar 2 Mental Health Services, Hamad Medical Corporation, Doha, Qatar International Journal of Clinical Pharmacy (2020) 42:110–123 https://doi.org/10.1007/s11096-019-00946-9 International Journal of Clinical Pharmacy (2020) 42:110–123 https://doi.org/10.1007/s11096-019-00946-9 RESEARCH ARTICLE Abstract Background Pharmacists in many developed countries have been granted prescribing authorities under what is known as “non-medical prescribing” or “pharmacist prescribing”. However, such prescribing privileges are not available in many developing countries. Objective The objective of this study was to determine the perspectives of future pharmacists (recent pharmacy graduates and pharmacy students) on pharmacist prescribing and its potential implementation in Qatar. Methods A convergent parallel mixed-methods design was used: (1) a cross-sectional survey using a pre-tested questionnaire and; (2) focus group discussions to allow for an in-depth understanding of the issue, with a focus on pharmacists prescribing competencies as well as barriers for its implementation. Main outcome measures Future pharmacists’ perspectives and atti- tudes towards pharmacist prescribing in Qatar. Results The majority of the respondents (94.4%) indicated awareness of the prescribing competency related to selecting treatment options. Furthermore, the majority (92.4%) believed that pharmacists should undergo prescribing training and accreditation before been legally allowed to prescribe, a point that was reiterated in the focus group discussions. Participants generally expressed support for collaborative and supplementary prescribing models when developing prescribing frameworks for Qatar. Four categories emerged under the theme barriers to implementation of pharmacist prescribing: lack of prescribing competency, pharmacist mindset, lack of accessibility to patient records and counseling rooms, and diversity of education and training background. Conclusion The majority of recent pharmacy gradu- ates and students were in favor of pharmacist prescribing been implemented in Qatar. However, a special training program was deemed necessary to qualify pharmacists to prescribe safely and effectively. Keywords Mixed-methods · Pharmacist prescribing · Pharmacy graduates · Pharmacy students · Qat Introduction In recent years, healthcare systems have been rapidly advancing with an increased focus on optimizing health- care quality, ensuring safety, and strengthening the provision of patient-centered care [1, 2]. In parallel with the globally evolving healthcare needs, there has been an apparent expan- sion of pharmacy practice beyond the traditional technical and dispensing paradigm [1–4]. Owing to pharmacists’ role Vol:.(123456789 1 3 l:.(123456789 1 3 International Journal of Clinical Pharmacy (2020) 42:110–123 111 pharmacist-interventions [23]. Likewise, much of the cur- rent Canadian literature pertaining to pharmacist prescribing activities has demonstrated significant evidence of improve- ments in similar clinical parameters [6, 24–29]. A number of stakeholders also reported that prescribing practices by pharmacists were considered safe and effective [6, 30]. Fur- thermore, physicians, patients, and NMPers have reported that NMP by nurses and pharmacists reduced the workload of MPers, improved the continuity of care, and increased job satisfaction and professional confidence [9, 10, 15, 30]. In addition, suboptimal medical prescribing has also been evident in the literature, suggesting the potential impact of NMP on reducing medication errors [31–36]. Studies assess- ing prescribing competencies of different healthcare students (e.g. medical, pharmacy, and nursing students) are scarce. One comparative study found that pharmacy students had better knowledge of basic pharmacology than medical stu- dents (77.0% vs. 68.2%; P < 0.001), yet both groups had sim- ilar knowledge of applied pharmacology (73.8% vs. 72.2%; P = 0.124) [35]. Another study also showed that pharmacy students had a significantly higher identification rate of pre- scription errors than that of medical students [36]. In addi- tion, evidence shows that pharmacists’ earlier involvement in the prescribing process may help optimize the use of medicines [37, 38]. pharmacist-interventions [23]. Likewise, much of the cur- rent Canadian literature pertaining to pharmacist prescribing activities has demonstrated significant evidence of improve- ments in similar clinical parameters [6, 24–29]. A number of stakeholders also reported that prescribing practices by pharmacists were considered safe and effective [6, 30]. Fur- thermore, physicians, patients, and NMPers have reported that NMP by nurses and pharmacists reduced the workload of MPers, improved the continuity of care, and increased job satisfaction and professional confidence [9, 10, 15, 30]. expansion and enhanced clinical training and professional expertise in evidence-based drug therapy management, they are considered as an integral part of healthcare teams [1, 2, 5, 6]. Introduction In addition to their involvement in medication therapy management, proactive patient counseling, and direct patient care, an evolving role for pharmacists is involvement in pre- scribing activities with varying degree of privileges [3–8]. Traditionally, the right to prescribe medicines was restricted to physicians and dentists only [8, 9]. However, over the past three decades, new policies and legislations have emerged to allow non-medical prescribing (NMP) [6, 10]. NMP is the legal right to prescribe medicines granted to healthcare pro- fessionals (HCPs) other than doctors and dentists, who have undergone a specified training and have attained appropriate competencies and qualifications to become prescribers [6, 8–11]. Pharmacists are among the range of other HCPs who have been given the right to prescribe in a number of devel- oped countries, including the United Kingdom, the United States of America, Canada, Australia, and New Zealand [6, 7, 10, 12–14]. However, the level of authority to prescribe may vary in different areas, in that legal restrictions could apply on who can prescribe medicines, what they can pre- scribe, and whether they are allowed to do so independently [6–9, 15–18]. For example, in Canada, Alberta was the first province to authorize independent pharmacist prescribing for registered pharmacists in 2007 [7]. Most pharmacists in other Canadian provinces pursued prescribing activities under col- laborative protocols before the legislation of such privileges across Canada in following years [7, 18–20]. Notably, the most common NMP models described in the literature include inde- pendent, dependent, and collaborative prescribing [6, 8, 10, 11, 17, 21–23]. The independent prescribing model grants a non- medical prescriber (NMPer) the sole responsibility of assess- ing, diagnosing, and making initial treatment decisions, while dependent NMPers are restricted in their activities by protocol/ formulary [8, 11]. Meanwhile, collaborative prescribers are granted the legal authority to prescribe medications under a collaborative relationship towards achieving a set of agreed patients’ outcomes [6, 8, 11]. i In addition, suboptimal medical prescribing has also been evident in the literature, suggesting the potential impact of NMP on reducing medication errors [31–36]. Studies assess- ing prescribing competencies of different healthcare students (e.g. medical, pharmacy, and nursing students) are scarce. One comparative study found that pharmacy students had better knowledge of basic pharmacology than medical stu- dents (77.0% vs. 68.2%; P < 0.001), yet both groups had sim- ilar knowledge of applied pharmacology (73.8% vs. 72.2%; P = 0.124) [35]. Study design and setting This study was conducted at the College of Pharmacy (CPH), QU. A convergent parallel mixed-methods design was used, where both quantitative and qualitative data were indepen- dently and concurrently collected to increase the validity and comprehensiveness of the data [41–43]. This would allow complementarity of both quantitative and qualitative method- ologies and an in-depth understanding of the research ques- tion. Below is a detailed account of the two components. Study sample and sampling technique All current pharmacy students in the CPH at QU (BSc, PharmD, and MSc in Pharmacy Practice) and recent phar- macy graduates (BSc graduates of the previous academic year who were not currently in the MSc or PharmD pro- gram at QU CPH) were invited to participate in the study (n = 146). All part-time PharmD and MSc students who did not receive their BSc at QU, in addition to recent graduates who were already employed, were excluded from the study. Universal sampling technique was followed in view of the low number of study population. Ethics approval The study received ethics approval from Qatar University (QU) Institutional Review Board (approval reference number: QU-IRB924-EA/18). The study did not involve any medical interventions or invasive procedures. Completing the web- based survey was an implied consent to participate in the cross-sectional survey. Informed consent forms were obtained from those who participated in focus group discussions. The questionnaire was divided into five sections: (1) Participants’ demographics; (2) Awareness regarding pharmacist prescribing and prescribing competencies; (3) Beliefs and views about prescriptive authority; (4) Perceptions regarding prescribing competencies covered in QU-CPH curriculum; (5) Barriers and facilitators to implementation of pharmacist prescribing in Qatar. All items were developed to adapt the culture and set- ting. The type of questions varied according to the purpose. Multiple answer questions were used for demographics sec- tion, barriers and facilitators. Close-ended questions such as (Yes/No/Not sure) were used for awareness section and items anchored on a five-point Likert scale were used for beliefs and views about prescribing authority and for perceptions regarding prescribing competencies covered in curriculum. Open ended questions were used as required. The question- naire underwent content validity by three pharmacy faculty members with expertise in pharmacy practice research and survey instrument development, specifically with experience in NMP research. Modifications were done to the draft of the questionnaire through an iterative process. Then, the ques- tionnaire was piloted using a sample of five recent gradu- ates, who were not eligible to participate in the study, to determine clarity, readability, and comprehensiveness of the developed items. Minor modifications were made before the final version of the questionnaire was made available. Aim of the study An anonymous, self-administered link using SurveyMonkey® online software (SurveyMonkey®, San Mateo, CA, USA) was designed and then circulated to participants via emails. The questionnaire used was con- structed based on the objectives of the study and through an extensive literature review. The three-main utilized key published literature related to prescribing were “Compe- tencies required to prescribe medicines” by National Pre- scribing Service at Australia and the Prescribing Com- petency Framework by Royal Pharmaceutical Society, in addition to the “Guide To Good Prescribing: A Practical Manual” by World Health Organization (WHO) [44–46]. Other pertinent studies around similar objectives were also used for developing the questionnaire [47–54].i The aim of this study was to determine, and explore in depth, the perspectives and attitudes of pharmacy students and recent pharmacy graduates towards pharmacist prescribing as an emerging role for pharmacists in Qatar. This study along with other studies from our larger project will pro- vide foundational basis for implementing an evidence-based practice, which addresses national health priorities that are aimed at improving efficiency in the healthcare system and improving access to medicines. Introduction Another study also showed that pharmacy students had a significantly higher identification rate of pre- scription errors than that of medical students [36]. In addi- tion, evidence shows that pharmacists’ earlier involvement in the prescribing process may help optimize the use of medicines [37, 38]. Currently, pharmacist prescribing is considered a novel area of practice in Qatar and the Middle East. Nonetheless, the need for expanding the scope of pharmacy practice/ services, including prescribing, corresponds with the new launch of Qatar’s National Health Strategy 2018–2022 [39]. Accordingly, it is important to determine the views, per- spectives, and experiences of all stakeholders—including pharmacy students—about prescribing competencies and the potential implementation of pharmacist prescribing in Qatar. Little is known about the perspectives and views of pharmacy students on prescribing competencies and imple- menting pharmacist prescribing. Only two studies were iden- tified that investigate pharmacy students’ perspectives on implementing NMP and acquisition of prescriptive authority [17, 40]. Despite the diversity in students’ views and expe- riences, students in both studies expressed their prepared- ness to undertake prescribing role. However, differing views among future pharmacists may arise due to the variability of laws, policies and priorities in different regions [17]. To our knowledge, there were no previous studies conducted in this regard in Qatar or in the Middle East. This study is part of a larger project (Pharmacist Prescribing in Qatar Project) in which the investigators look at various stakehold- ers (including pharmacists, pharmacy educators, pharmacy students and recent graduates, academic leaders in health professional education, health policymakers etc.) who may have a potential role in the development and implementation of pharmacist prescribing in the State of Qatar. Essentially, the pharmacist prescribing initiative is aimed at improving the quality and continuity of patient care, improving medication management, improving patients’ access to medicines and healthcare, saving time for patients and medical practitioners, and improving the utilization of resources by making better use of professional skills and expertise of the pharmacist [4, 9, 10, 15]. A recent system- atic review of 45 studies, demonstrated positive outcomes in the management of acute and chronic diseases in favor of pharmacists and nurses NMP against medical prescrib- ers (MPers) [23]. Such outcomes included improvements in lipid profiles, glycated hemoglobin, and blood pres- sure levels. Additionally, the meta-analysis of 14 studies showed that 85% of patients were generally satisfied with 1 3 International Journal of Clinical Pharmacy (2020) 42:110–123 112 Sampling and recruitment Students and recent graduates who participated in the quan- titative phase of the study were invited to take part in focus group discussions (FGDs) to allow for an in-depth under- standing of the issue, with a focus on pharmacists prescrib- ing competencies and inclusion of content in pharmacy curriculum or development of continuing professional 1 3 International Journal of Clinical Pharmacy (2020) 42:110–123 113 Table 1 Future pharmacists’ demographic and academic characteris- tics (N = 105) n, number of respondents Parameters n (%) Age 18–20 years 27 (25.7) 21–23 years 54 (51.4) 24–26 years 24 (22.9) Nationality Qatari 10 (9.5) Egyptian 39 (37.1) Sudanese 16 (15.2) Jordanian 10 (9.5) Palestinian 5 (4.8) Syrian 7 (6.7) Others 18 (17.1) Current year in pharmacy college First professional year (P1) 21 (20) Second professional year (P2) 19 (18.1) Third professional year (P3) 17 (16.2) Fourth professional year (P4) 20 (19.0) Recent BSc graduates of the previous year 28 (26.7) Number of clinical rotations completed so far during BSc and/or PharmD 0 40 (38.1) 1 20 (19.0) 2 1 (1.0) 3 1 (1.0) 4 1 (1.0) 5 0 6 29 (27.6) More than 6 13 (12.4) development courses related to pharmacist prescribing. Each focus group session compromised 6–7 participants and lasted for about 60–90 min. The concept of theoretical saturation was followed. After four focus groups, no novel themes were emerged, and saturation was judged to be reached. Data collection A topic guide for the focus group discussions was developed based on the same literature review used in Phase 1. The guide was developed by two researchers and tested for validity by five other research members. Each focus group session was audiotaped using a digital audio recorder. Each session was transcribed verbatim with consideration to non-verbal expres- sions. A thematic approach of data analysis was used, and a framework was generated which allowed for structuring, labe- ling and defining data. Each phrase was coded with a code that reflects the meaning of the passage. Similar phrases, passages and ideas were sorted together under the same code and all these codes were used to generate themes. Data analysis For quantitative phase, statistical analyses were performed using Statistical Package for Social Sciences, version 24 (IBM SPSS® Statistics for Windows; IBM Corp, Armonk, New York, USA). Frequencies and percentages were used to summarize the responses generated. For qualitative phase, thematic content analysis was used to structure the information derived from focus group discussions. Coding was done manually by three investigators with experience in qualitative research. Participants’ demographic characteristics providing information to other health professionals to ensure that the treatment plan is implemented safely and effectively)a 95 (91.3) 7 (6.7) 2 (1.9) Monitoring and reviewing (e.g. obtaining and interpreting information to decide whether the therapeutic goals have been achieved whether to continue/stop treatment or refer the person to another health professional for further assessment) 94 (89.5) 4 (3.8) 7 (6.7) Prescribing safely (e.g.: know and report prescribing errors, minimize risks, keep up-to-date with safety concerns) 93 (88.6) 6 (5.7) 6 (5.7) Prescribing professionally (e.g. practicing in accordance with the relevant legislative, regulatory, professional, and organizational frameworks and applying quality use of medicines principles) 65 (65.7) 15 (14.3) 25 (23.8) Improving prescribing practice (e.g. working to continually improve prescribing practice by reflecting and act- ing upon feedbacks, discussions, unsafe prescribing and understanding available tools to improve prescrib- ing) 69 (65.7) 15 (14.3) 21 (20) Prescribing as part of a team (e.g.: communicate and collaborate effectively with the person and other health professionals)a 81 (77.9) 10 (9.6) 13 (12.5) Fig. 1 Future pharmacists’ awareness of international prescribing models 7.60% 4.80% 2.90% 1.90% 5.70% 5.70% 5.70% 3.80% Independent Prescribing Dependent Prescribing model: Supplementary Prescribing Dependent Prescribing model: Prescribing by Protocols Dependent Prescribing model: Patients Group Direction Dependent Prescribing model: Prescribing by formulary Dependent Prescribing model: Prescribing by patient referral Dependent Prescribing model: Repeat Prescribing Collaborative Prescribing Models Models of Prescribing Table 2 Students’ and recent graduates’ awareness of prescribing competencies (N = 105) Statements Table 2 Students’ and recent graduates’ awareness of prescribing competencies (N = 105) Table 2 Students’ and recent graduates’ awareness of prescribing competencies (N = 105) a Missing data = 1 Statements n (%) Yes No Not sure Assessing the patient (e.g. obtaining information to understand a person’s clinical needs, performing a com- prehensive medicines assessment, generating and exploring possible diagnosis) 94 (89.5) 4 (3.8) 7 (6.7) Considering treatment options (e.g. identifying and discussing appropriate, safe, effective, and evidence-based treatments for the patient) 100 (95.2) 4 (3.8) 1 (1.0) Reaching a shared decision (e.g. negotiating therapeutic goals, reaching agreement about medicines to treat the person’s condition, and tailoring the treatment plan to meet the needs of the person)a 94 (90.4) 7 (6.7) 3 (2.9) Prescribing medicine (e.g. Participants’ demographic characteristics More than two-thirds of the participants (n = 76, 72.4%) had not observed the role of the pharmacist as a prescriber (either independently or through a protocol) in Qatar. Only 31 (29.5%) respondents expressed awareness (i.e. knowing and understand- ing) of at least one international prescribing competency frame- work. However, more than 66–95% of them expressed aware- ness of individual competency areas listed in the international framework as shown in Table 2. About 38% and 34% of the respondents were unsure or unaware of the prescribing compe- tency related to prescribing professionally and improving pre- scribing practice, respectively. Only 8.6% of them were aware of the different models of NMP that are being implemented inter- nationally. The most known prescribing model pointed by the participants was independent prescribing. Figure 1 illustrates the respondents’ awareness of international prescribing models. Of the 146 eligible students and recent graduates who were invited to participate in the study, 105 (72%) completed the online survey. About 28 (26.7%) were recent graduates, while the rest were students across all professional years. As shown in Table 1, around half of them (n = 54, 51.4%) were 21–23 years old. The majority of the participants (n = 55, 52.3%) were from Egypt and Sudan. About 38% of the participants have not had any experiential training (i.e. clinical rotations). Further details about the demographics of the participants are provided in Table 1. 1 1 3 3 International Journal of Clinical Pharmacy (2020) 42:110–123 114 114 International Journal of Clinical Pharmacy (2020) 42:110–123 Table 2 Students’ and recent graduates’ awareness of prescribing competencies (N = 105) a Missing data = 1 Statements n (%) Yes No Not sure Assessing the patient (e.g. obtaining information to understand a person’s clinical needs, performing a com- prehensive medicines assessment, generating and exploring possible diagnosis) 94 (89.5) 4 (3.8) 7 (6.7) Considering treatment options (e.g. identifying and discussing appropriate, safe, effective, and evidence-based treatments for the patient) 100 (95.2) 4 (3.8) 1 (1.0) Reaching a shared decision (e.g. negotiating therapeutic goals, reaching agreement about medicines to treat the person’s condition, and tailoring the treatment plan to meet the needs of the person)a 94 (90.4) 7 (6.7) 3 (2.9) Prescribing medicine (e.g. prescribing with adequate, up-to-date awareness of medication actions, indications, dose, contraindications, interactions, cautions, and unwanted effects; prescribing within relevant frameworks for medicines use… etc.) 74 (70.5) 12 (11.4) 19 (18.1) Providing information (e.g. Future pharmacists’ perceptions regarding prescribing competencies covered in the current pharmacy curriculum with reference to WHO standards With regards to the current curricula in the CPH, partici- pants had differing perceptions regarding their preparedness Table 3 Beliefs and views of future pharmacists about prescriptive authority and implementation of pharmacist prescribing in Qatar (N = 105) Statements n (%) Disagree Neutral Agree 1. I think pharmacist can accurately assess and diagnose patients within their competencea 11 (10.6) 26 (25) 67 (64.4) 2. Overall, I believe pharmacists can accurately choose and initiate the optimal drug therapy for a patient’s diagnosis 1 (1) 12 (11.4) 92 (87.6) 3. I believe pharmacists can accurately adjust a medication regimen to improve patient outcomes 0 5 (4.8) 100 (95.2) 4. I believe pharmacists should have prescribing training and accreditation or certification before being legally allowed to prescribe medicines within their competence 3 (2.9) 5 (4.8) 97 (92.4) 5. I believe all pharmacists should have prescriptive authority (i.e. legal authority to prescribe certain medi- cines) 22 (21) 17 (16.2) 66 (62.8) 6. Pharmacists are able to prescribe safely and effectively 2 (1.9) 11 (10.5) 92 (87.7) 7. Pharmacists are able to prescribe cost-effectively 6 (5.8) 10 (9.5) 89 (84.7) 8. Prescribing by pharmacists is likely to be acceptable to patients 20 (19.1) 25 (23.8) 60 (57.1) 9. Physicians are likely to be in favor of prescribing by pharmacists 57 (54.2) 22 (21) 26 (24.7) 10. Prescribing by pharmacists may potentially reduce medication errors in practicea 4 (3.8) 17 (16.3) 83 (79.8) 11. Pharmacist prescribing will increase better utilization of pharmacist’s skills and knowledge 3 (2.9) 5 (4.8) 97 (92.4) 12. Pharmacist prescribing will lead to improved patient access to medicines 3 (2.9) 14 (13.3) 88 (83.8) 13. Patients accessing prescribing by pharmacists will have better continuity of care 2 (2) 16 (15.2) 87 (82.9) 14. Prescribing authority for pharmacists will increase job satisfactions 3 (2.9) 8 (7.6) 94 (89.6) 15. Prescribing authority for pharmacists will increase self-confidence 1 (1) 5 (4.8) 99 (94.3) 16. Prescribing by pharmacists will reduce physician’s workload so they focus on more acute patients’ cases 5 (4.8) 16 (15.2) 84 (80) 17. Pharmacist prescribers must have access to the information in physician’s medical notes prior to prescrib- ing 0 3 (2.9) 102 (97.1) 18. Pharmacist prescribers must be able to record prescribing actions in medical recordsa 0 4 (3.8) 100 (96.2) 19. Barriers and facilitators to implementation of pharmacist prescribing in Qatar Figure 2 illustrates the perceived barriers to the implemen- tation of pharmacist prescribing in Qatar. Legal rights and restrictions to prescribe was the most commonly reported barrier (75.2%). This was followed by lack of collaborative practice (63.8%), lack of private consultation rooms (56.2%) and lack of access to medical records (56%). Lack of skills in monitoring drug therapy (17.1%) and pharmacist not inter- ested in undertaking the new prescribing role were the least perceived barriers (19%). With regards to the facilitators, inter- professional collaboration was the most commonly reported facilitator (73.3%), followed by advancements in pharmacy practice and pharmacist’s role (72.4%) as shown in Fig. 3. Beliefs and views of future pharmacists about prescriptive authority and implementations of pharmacist prescribing in Qatar with reference to WHO prescribing competency standards as shown in Table 4. Participants expressed preparedness to educate and provide instructions about the use of the prescribed medication (80.2%). About 82% of the partici- pants agreed or strongly agreed to the statement: “Overall, I believe that I need special training and clinical experience on prescribing to be able to prescribe medicines”. More than 90% of the participants believed that phar- macists can accurately adjust medication regimens to improve patient outcomes and that they should have an accredited prescribing training before being legally allowed to prescribe medicines. However, around one- fifth of the participants disagreed that pharmacist pre- scribing is likely to be accepted by patients. Overall, 57–97% of the participants agreed to all the statements in Table 3, except for one statement. There was less agree- ment to the statement “Physicians are likely to be in favor of prescribing by pharmacists”, where only 24.7% of the participants agreed to the statement. Participants’ demographic characteristics prescribing with adequate, up-to-date awareness of medication actions, indications, dose, contraindications, interactions, cautions, and unwanted effects; prescribing within relevant frameworks for medicines use… etc.) 74 (70.5) 12 (11.4) 19 (18.1) Providing information (e.g. providing information to other health professionals to ensure that the treatment plan is implemented safely and effectively)a 95 (91.3) 7 (6.7) 2 (1.9) Monitoring and reviewing (e.g. obtaining and interpreting information to decide whether the therapeutic goals have been achieved whether to continue/stop treatment or refer the person to another health professional for further assessment) 94 (89.5) 4 (3.8) 7 (6.7) Prescribing safely (e.g.: know and report prescribing errors, minimize risks, keep up-to-date with safety concerns) 93 (88.6) 6 (5.7) 6 (5.7) Prescribing professionally (e.g. practicing in accordance with the relevant legislative, regulatory, professional, and organizational frameworks and applying quality use of medicines principles) 65 (65.7) 15 (14.3) 25 (23.8) Improving prescribing practice (e.g. working to continually improve prescribing practice by reflecting and act- ing upon feedbacks, discussions, unsafe prescribing and understanding available tools to improve prescrib- ing) 69 (65.7) 15 (14.3) 21 (20) Prescribing as part of a team (e.g.: communicate and collaborate effectively with the person and other health professionals)a 81 (77.9) 10 (9.6) 13 (12.5) 7.60% 4.80% 2.90% 1.90% 5.70% 5.70% 5.70% 3.80% Independent Prescribing Dependent Prescribing model: Supplementary Prescribing Dependent Prescribing model: Prescribing by Protocols Dependent Prescribing model: Patients Group Direction Dependent Prescribing model: Prescribing by formulary Dependent Prescribing model: Prescribing by patient referral Dependent Prescribing model: Repeat Prescribing Collaborative Prescribing Models 0% 2% 4% 6% 8% Models of Prescribing Percentage Fig. 1 Future pharmacists’ awareness of international prescribing models 1 3 3 115 International Journal of Clinical Pharmacy (2020) 42:110–123 Beliefs and views of future pharmacists about prescriptive authority and implementations of pharmacist prescribing in Qatar Future pharmacists’ perceptions regarding prescribing competencies covered in the current pharmacy curriculum with reference to WHO standards 2 Future pharmacists’ per- ceived barriers to the implemen- tation of pharmacist prescribing in Qatar 23.8 63.8 46.7 40 54.3 17.1 49.5 28.6 19 42.9 75.2 56.2 56 28.6 0 10 20 30 40 50 60 70 80 Problems in implemenng care plans Lack of collaborave pracce Lack of employer support Increased workload Inadequate training in paent assessment and diagnosis Lack of skills in monitoring drug therapy Inadeqate training on prescribing competencies Lack of self-conﬁdence Pharmacists are not interested in undertaking the new role… Lack of acceptance by paents Legal rights and restricons to prescribe Lack of private consultaon room Lack of access to paent medical records Lack of me Percentage (%) Barriers Table 4 Future pharmacists’ perceptions of prescribing competencies covered in the current pharmacy curriculum with reference to WHO pre- scribing competency standards (N = 101) Competency standard n (%) sts’ per- mplemen- escribing 23.8 63.8 46.7 40 54.3 17.1 49.5 28.6 19 42.9 75.2 56.2 56 28.6 0 10 20 30 40 50 60 70 80 Problems in implemenng care plans Lack of collaborave pracce Lack of employer support Increased workload Inadequate training in paent assessment and diagnosis Lack of skills in monitoring drug therapy Inadeqate training on prescribing competencies Lack of self-conﬁdence Pharmacists are not interested in undertaking the new role… Lack of acceptance by paents Legal rights and restricons to prescribe Lack of private consultaon room Lack of access to paent medical records Lack of me Percentage (%) Barriers Future pharmacists’ perceptions regarding prescribing competencies covered in the current pharmacy curriculum with reference to WHO standards I feel it is my professional duty and obligation to become a pharmacist prescriber upon graduation 8 (7.6) 14 (13.3) 83 (79.1) 20. Overall, I feel confident in my ability to become a pharmacist prescriber upon graduation 4 (3.9) 18 (17.1) 83 (79) a N = 104 a N = 104 1 International Journal of Clinical Pharmacy (2020) 42:110–123 116 Focus group discussions Participants’ demographics and themes Twenty-four future pharmacists consented to participate in Theme 1: benefits of pharmacist prescribing Three categories emerged under the “benefits of pharma- cist prescribing” theme. These are: benefits to HCPs and health care system (HCS), benefits to pharmacy profes- i d h i d b fi i l i d Table 4 Future pharmacists’ perceptions of prescribing competencies covered in the current pharmacy curriculum with reference to WHO pre- scribing competency standards (N = 101) N = 101 (missing four responses in this section) Competency standard n (%) Unprepared Somewhat unprepared Neutral Somewhat prepared Prepared Assessing and defining patient’s problems (e.g. disease or disorder, sign of underlying disease, side effect of drugs, non-adherence to treatment) 4 (4) 5 (5) 10 (9.9) 35 (34.7) 47 (46.5) Specifying therapeutic goals 5 (5) 1 (1) 4 (4) 24 (23.8) 67 (66.3) Specifying alternative treatment (pharmacologic and non-pharmacologic) 4 (4) 1 (1) 6 (5.9) 16 (15.8) 74 (73.3) Choosing a drug that is effective (by checking indication and conveni- ence), safe (by checking contraindications, interactions and high-risk group) with low cost 2 (2) 3 (3) 8 (7.9) 18 (17.8) 70 (69.3) Verifying suitable dose, route, dosage form, frequency and duration of drug for the patient 2 (2) 4 (4) 6 (5.9) 22 (21.8) 67 (66.3) Writing a drug prescription independently 7 (6.9) 12 (11.9) 18 (17.8) 29 (28.7) 35 (34.7) Educating and providing instructions about the use of the prescribed medication 2 (2) 2 (2) 6 (5.9) 10 (9.9) 81 (80.2) Monitoring the outcome of drug therapy 3 (3) 4 (4) 8 (7.9) 26 (25.7) 60 (59.4) Reviewing/altering prescription in the light of further investigations 3 (3) 6 (5.9) 13 (12) 20 (19.8) 59 (58.4) Fig. Participants’ demographics and themes Three categories emerged under the “benefits of pharma- cist prescribing” theme. These are: benefits to HCPs and health care system (HCS), benefits to pharmacy profes- sion and pharmacists, and benefits to patients as explained below.f Twenty-four future pharmacists consented to participate in the focus group discussions (FGDs). There were 4, 8, 3, 2 and 7 participants from P1, P2, P3, P4 and recent gradu- ates, respectively. The mean (SD) duration for FGDs was 73 ± 17.3 min. Six main themes emerged from the FGDs. Table 5 represents a summary of the identified themes and their categories. Reducing burden, balancing tasks and saving efforts, reducing errors, prioritization of cases (minor vs. compli- cated) and better safe prescribing were all recognized as advantages for HCPs and HCS. 1 3 International Journal of Clinical Pharmacy (2020) 42:110–123 117 International Journal of Clinical Pharmacy (2020) 42:110–123 Fig. 3 Future pharmacists’ perceived facilitators to the implementation of pharmacist prescribing in Qatar 73.3 56.2 54.3 58.1 63.8 64.8 63.8 72.4 56.2 53.3 0 10 20 30 40 50 60 70 80 Interprofessional collaboraon Prescribers’ enthusiasm Individualized professional development… Understanding roles of each health care professional Access to appropriate informaon technology (IT)… The use of formularies and guidelines Pharmacist interest Advancement in pharmacy pracce and … Having appropriate knowledge in non-medical… Having appropriate skills in non-medical prescribing Percentage Facilitators Table 5 Identified themes and subthemes/categories that emerged during focus group discussions Themes Categories Benefits of pharmacist prescribing Benefits to healthcare providers and system Benefits to pharmacy profession Benefits to patients Barriers to implementation of pharmacist prescribing Pharmacist incompetency Pharmacist mindset Lack of accessibility and support Pharmacist burden Diversity of education and training background Level and model of prescribing Knowledge of prescribing models Prescribing setting and restrictions Preferred models of prescribing Facilitators of pharmacist prescribing in Qatar Healthcare provider-related factors Pharmacist and pharmacy profession related-factors Patient-related factors Others: including governmental factors Pharmacy curriculum Participant’s awareness of prescribing guidelines Characteristics of current pharmacy program Gaps in the current pharmacy program Implementation process Characteristics of a training program Ways of implementation Fig. Participants’ demographics and themes Better care provided for patients and again you know community pharmacies are really spread and they can find near to them so they can easily reach (FGD1, X1) Better care provided for patients and again you know community pharmacies are really spread and they can find near to them so they can easily reach (FGD1, X1) I think that here in Qatar we have different pharmacy practitioners from different universities, not all of them have gone through the same thing (FGD 2, X5) Theme 3: level and model of prescribing Because if we just suddenly start with independent, it is going to make the stigma more and relationship will be more complicated between health care providers and pharmacists (FGD3, X4) Because of the training … physicians are taking the whole load … But I don’t think pharmacists they have this mentality that can go actually diagnose and pre- scribe … (FGD1, X2) I think we can go with supplementary, make people who are independent prescribers be more adapt to the idea that is not all on you, we can share the responsibil- ity and if they get to the idea or the rationale… If that could be installed on their heads, we can go into more collaborative practices (FGD4, X3) Lack of accessibility to patient records and counseling rooms, inability to order laboratory investigations, lack of support from physicians, public, and patients were also perceived barriers related to accessibility that support this subtheme. …Lack of facilities also, such as physical structure of some settings. Counseling rooms are not available (FGD2, X3) Theme 3: level and model of prescribing Five categories emerged under the theme “barriers to imple- mentation of pharmacist prescribing”, including: lack of pharmacist competency in prescribing, pharmacist mind- set, lack of accessibility and support, pharmacist burden and diversity of education and training background. Knowledge of prescribing models, prescribing setting and restrictions, and preferred models of prescribing were the categories that emerged under this theme. Few participants were aware of the models implemented internationally. In Qatar, they have pointed that one known model is the collaborative agreement protocol that is being applied in the Heart Hospital, one of the Hamad Medical Corporation hospitals in Qatar. Lack of information and skills on diagnosis, lack of proper training and negligence of responsibilities are all incompetency of pharmacists highlighted by the participants. In addition, some participants stated that some pharmacists are incompetent when providing counseling to patients. [Pharmacist] can only change warfarin dose to increase or decrease… The physician will diagnose and tell that this patient is having heart failure…,and then the phar- macist will suggest the dose of warfarin … and adjust according to INR (FGD4, X5) The pharmacists never looked at me and asked … he then gave me the medications. So, I mean this can be a disaster if we give such person the authority to diag- nose and prescribe. (FGD1, X3) For example, people from our college you see them they want to counsel and educate patients. However, you see people from outside who don’t have that? (FGD2, X4) In relation to which model of prescribing suits the Qatar context, the majority agreed that the supplementary pre- scribing model would be the most suitable. Few interviewees suggested gradual implementation of pharmacist prescribing in Qatar. Additionally, pharmacist mindset has been perceived as a barrier to implementation of pharmacist prescribing. Dis- inclination, fear of additional load, inflexible habit of mind by pharmacists, and having a traditional social perception on the pharmacy profession were recognized as barriers that may hinder pharmacist prescribing. I think supplementary [prescribing] would work bet- ter. I think the idea of having a shared care plan is a nice way to ease the pharmacists’ role in prescribing. Participants’ demographics and themes 3 Future pharmacists’ perceived facilitators to the implementation of pharmacist prescribing in Qatar Table 5 Identified themes and subthemes/categories that emerged during focus group discussions Themes Categories Benefits of pharmacist prescribing Benefits to healthcare providers and system Benefits to pharmacy profession Benefits to patients Barriers to implementation of pharmacist prescribing Pharmacist incompetency Pharmacist mindset Lack of accessibility and support Pharmacist burden Diversity of education and training background Level and model of prescribing Knowledge of prescribing models Prescribing setting and restrictions Preferred models of prescribing Facilitators of pharmacist prescribing in Qatar Healthcare provider-related factors Pharmacist and pharmacy profession related-factors Patient-related factors Others: including governmental factors Pharmacy curriculum Participant’s awareness of prescribing guidelines Characteristics of current pharmacy program Gaps in the current pharmacy program Implementation process Characteristics of a training program Ways of implementation Pharmacy curriculum Implementation process Many pharmacists have the ability to do minor diag- nosis for some minor illnesses… So this will decrease the workload on the physicians (FGD2, X4) professional development. In addition, it will be an oppor- tunity for pharmacists to apply their knowledge. I think the huge advantage is that the public would be more aware of our role. If you are proving this opportunity, the public will view pharmacy like not a dispensing machine … the expectations will be much higher (FGD 4, X3) … If pharmacists had more control over the prescrib- ing process, these errors would not happen … So that would really keep the health centers to focus on patients who really need physicians (FGD 1, X3) The participants have expressed that prescribing by pharmacist is an advantage for the profession and pharma- cists themselves. Pharmacists will have better job satisfac- tion, enhanced confidence, better public image and better Regarding benefits to patients, participants argued that pharmacist prescribing will save time for patients, offer them convenient and better care, and offer easy accessibility to HCS, 1 3 118 International Journal of Clinical Pharmacy (2020) 42:110–123 background, and variation in the role of pharmacists in com- munity versus hospital setting were recognized. 1 3 Theme 6: implementation process Additionally, facilitators of prescribing by pharmacists included pharmacy profession and pharmacists related fac- tors such as capacity building and manpower development, providing training programs, authorizations to access patient information, and availability of competent pharmacists. Two categories have emerged during the discussions related to the implementation of pharmacist prescribing in Qatar: (1) characteristics of training programs and; (2) the imple- mentation approaches. A group of participants suggested that integrating prescribing in their undergraduate courses would help in implementing pharmacist prescribing in Qatar. Others agreed that it has to be a standalone program that is certified and accredited to allow graduates to become prescribers. They [Pharmacists] are available so why not to use the manpower that you have (FGD1, X3) With the college focusing on prescribing, the pharma- cists will not only have the knowledge for prescrib- ing but also will grow accustomed to this mentality (FGD1, X3) They [Pharmacists] are available so why not to use the manpower that you have (FGD1, X3) With the college focusing on prescribing, the pharma- cists will not only have the knowledge for prescrib- ing but also will grow accustomed to this mentality (FGD1, X3) For QU students, definitely integration, because I think we cover a lot of aspects of prescribing in other courses. If we integrated into one of the courses, it will be less load to the student but for outside of QU [graduates] definitely should be a course or training (FGD4, X3) Theme 5: pharmacy curriculum Regarding participant’s awareness of prescribing guidelines, most of the participants were not aware. However, some of them believed that they heard about them, but never went through them in details. In addition, they have highlighted the characteristics of the pharmacy program at QU and stated that it is comprehensive, provide them with crucial knowledge and skills in a stepwise approach. Also, the pro- gram focuses on evidence-based practice which helps them in future as prescribers. For me, I don’t think the pharmacists really need a course, prescribing is just knowing the knowledge and applying it for choosing the drug. I don’t think it needs a whole course (FGD1, x2) I think it is being done already in other countries like diploma so I believe we need to have the same thing here for six months for examples. (FGD2, X3) I believe one of the important skills that we learn in the undergraduate program is that we learn how to search and how to use guidelines and how to follow evidence- based practice. So that’s why maybe it is easier for us to know how to prescribe (FGD2, X3) Theme 4: facilitators of pharmacist prescribing Four subthemes emerged as facilitators of pharmacist pre- scribing. These are factors related to: health care providers, pharmacist and pharmacy profession, patients, and others including governmental factors. The patients and the physicians they will be resistant to the expansion of the role of pharmacists because they may feel threatened. (FGD4, X4) Participants have indicated that support should come by implementing regulations and policies and having a Diversity of education and training background was an important subtheme under the barriers. Practice incon- sistency, having pharmacist from different educational 1 3 1 3 International Journal of Clinical Pharmacy (2020) 42:110–123 119 formal professional body (association) that speaks up for pharmacists. I think we have gaps in terms of the materials related to diagnosing. We don’t take enough courses related to diagnosis of different diseases. We have only patient assessment. We take two courses and they are not enough. They are focused to certain things, but not to all related to human beings and also for the prescrip- tion, we didn’t take what a legal prescription should look like. (FGD2, X4) I think we should have a pharmacy association or something or someone who can overlook the pharma- cists and show that everything [including prescribing implementation] is going smoothly…. (FGD2, X8) I think stakeholders and the governments, since they put rules and regulations, they should be the main sup- port for the implementation of prescribing (FGD2, X6) Discussion The current study aimed to investigate the awareness, views, and attitudes of future pharmacists (pharmacy students and recent graduates) regarding the potential implementation of pharmacist prescribing in Qatar. Gen- erally, the study participants had a poor awareness of internationally-recognized prescribing models. Never- theless, the participants indicated that the pharmacy cur- riculum at QU-CPH has provided them with sufficient set of skills and knowledge pertaining to prescribing com- petencies. Yet, some participants highlighted the short- fall of the curriculum in detailing significant competency areas necessary to assume future prescribing roles. Such domains include diagnosing disease conditions and writ- ing a prescription independently. Meanwhile, most study Furthermore, participants have highlighted that graduates from the PharmD program at QU are competent to prescribe. I found them [QU PharmD graduates] very competent to prescribe and even they suggest the chemotherapy protocols (FGD2, X5) However, some gaps in the pharmacy curriculum were reported. These included having some courses that are theo- retical more than practical, insufficient courses related to patient assessment and diagnosis, and the need for additional preparatory courses that focus on prescribing. 1 3 3 International Journal of Clinical Pharmacy (2020) 42:110–123 120 participants felt prepared to undertake roles in accordance with the prescribing competencies covered in the current pharmacy curriculum of the CPH at QU with reference to the WHO prescribing competency standards. Consistent with these findings, a study conducted in the UK demon- strated significant improvements in prescribing assessment scores (P = 0.007) and prescribing confidence (P = 0.0002) among final-year medical students following a doctor- and pharmacist-led prescribing teaching program, which was aimed at improving practical prescribing skills [55]. Pre- vious studies among pharmacy students from Canada and Australia have also reported positive attitudes and con- fidence in undertaking pharmacist prescribing role [17]. culture in the undergraduate curriculum [17]. Interestingly, the undergraduate pharmacy curriculum at the CPH in QU covers such specific courses as ‘Patient Assessment Labora- tory’ and ‘Interpretation of Clinical Laboratory Data’. Further, the study participants disagreed that physicians will be in favor of pharmacist prescribing. One of the most common barriers highlighted by our focus group participants was the potential lack of support from other stakeholders such as physicians. According to the literature, the main barrier to implementing NMP is perceived to be the fear of physicians’ resistance due to impression of overlapping duties [56, 68–72]. Discussion Pharmacy students from other studies have also indicated that physicians’ backlash and blurring of the profession may influence the willingness of pharmacists to undertake prescribing roles [17, 40]. Further, it has been suggested that adopting a clearly defined scope of practice and recognizing the boundaries and limitations of the phar- macy profession could avert the issue of overlapping duties with physicians and other HCPs [17]. Another study that explored the perspectives of pharmacy students in Qatar on interprofessional education and collaborative practice, reported that pharmacy students were least confident about their professional identity [73]. This was attributed to lack of role models, existence of power and hierarchy between healthcare team members, and resistance to the evolving role of the pharmacists, especially by physicians [73].i i Overall, most of the current study participants have high- lighted the necessity of establishing special prescribing training programs to facilitate the acquisition of pharmacist prescriptive authority. Corresponding remarks pertaining to NMP implementation were reported in other countries by practicing pharmacists and other HCPs [12, 16, 30, 56–63]. Significant improvements in pharmacists’ knowledge were demonstrated through a competency assessment adminis- tered by the Saskatchewan government in Canada for phar- macists who intend to undertake independent prescribing roles for emergency contraption (ECP) services upon the completion of a specific training program for ECP prescrib- ing [64]. It has also been reported that improvements in the educational structure could guarantee the generation of confident and skillful professionals instead of fearful and risk averting ones [12, 17, 56, 57]. Professional confidence could also be essential for proper practice implementation [65–67]. For instance, given the fact that many qualified NMPers do not carry out prescribing activities, the outcomes of implementing NMP are not yet clear. Thus, strategies of implementing NMP at different settings may neither be well evaluated nor a reliable reflection of real-world practice [11, 16, 17, 60–63, 65–67]. The current study participants indicated the significance of receiving support through the amendment of laws and policies in favor of NMP. They have also identified interprofessional collaboration as a facilitator for implementing pharmacist prescribing. Similar perspectives and views were reported in the literature, whereby pharmacy students indicated the need for a well-established interprofessional experience aimed to increase the acceptance of pharmacists’ activities by other HCPs [17, 40]. Conclusion g 11. Stewart D, Jebara T, Cunningham S, Awaisu A, Pallivalapila A, MacLure K. Future perspectives on nonmedical prescribing. Ther Adv Drug Saf. 2017;8(6):183–97.f The majority future pharmacy practitioners in Qatar were in support of implementing pharmacist prescribing. In addition, they have demonstrated their willingness to undertake pre- scribing roles in their future practices, particularly under a col- laborative practice agreement. However, most participants rec- ognized the need to establish focused pharmacist-prescribing training programs and to include relevant content in pharmacy curricula to further improve pharmacists’ practical prescribing skills prior to implementation. To further broaden the findings of the current study, future studies should investigate the per- spectives of other healthcare students (e.g. medical, nursing, and dental students) on pharmacist prescribing and NMP in Qatar. g 12. Raghunandan R, Tordoff J, Smith A. Non-medical prescribing in New Zealand: an overview of prescribing rights, service delivery models and training. Ther Adv Drug Saf. 2017;8(11):349–60. 13. Tonna AP, Stewart D, McCaig D. An international overview of some pharmacist prescribing models. J Malta Coll Pharm Pract. 2008;14:20–6. 14. Hoti K, Hughes J, Sunderland B. Training of pharmacist prescrib- ers. Int J Pharm Pract. 2014;22:38–46. 15. Stewart D, MacLure K, George J. Educating non-medical pre- scribers. Br J Clin Pharmacol. 2012;74:662–7. 16. Tonna AP, Stewart D, West B, McCaig D. Pharmacist prescribing in the UK: a literature review of current practice and research. J Clin Pharm Ther. 2007;32(6):545–56. 17. Charrois T, Rosenthal M, Hoti K, Hughes C. Pharmacy student perceptions of pharmacist prescribing: a comparison study. Phar- macy. 2013;1(2):237–47. y 18. Amirault P. Update on pharmacists prescribing. Pharm Post. 2006;14:6–8. Acknowledgements Open Access funding provided by the Qatar National Library. This research was funded by QU under the National Capacity Building Research Grant (QUCP-CPH-2018/2019-2). 19. Hughes C, Makowsky M, Sadowski C, Schindel T, Yuksel N, Guirguis L. What prescribing means to pharmacists: a qualita- tive exploration of practising pharmacists in Alberta. Int J Pharm Pract. 2013;22(4):283–91. Funding The funders had no role in the design, planning and imple- mentation of the project. 20. Law MR, Ma T, Fisher J, Sketris IS. Independent pharmacist pre- scribing in Canada. Can Pharm J. 2012;145:17–23. Conflicts of interest The authors declare no conflict of interest in this study. Conflicts of interest The authors declare no conflict of interest in this study. 21. Emmerton L, Marriott J, Bessell T, Nissen L, Dean L. Discussion Consistent with other research studies, most of the participants in this study considered supplementary and collaborative prescribing as the most suitable models to be implemented in Qatar [10, 17, 23, 40]. Particularly, future pharmacists believed that a gradual acquisition of prescrip- tive authority by pharmacists could help establish a trust culture between pharmacists and other HCPs and evaluate possible areas of improvements in pharmacists’ prescribing competencies. Differing views have emerged in the current study about applying training or education related to prescribing qualifi- cations. While some participants were in favor of incorporat- ing practical prescribing courses within the undergraduate degree curriculum, particularly those aimed at improving assessment and diagnosis skills, other participants were more inclined to establishing a standalone certified post- graduate program focused on NMP. Consistent with this, varying perspectives were reported in the literature regard- ing the inauguration of educational structures for the pur- pose of obtaining a NMP qualification [17, 57]. According to a 2013 study [17], students from an Australian university believed that a NMP qualification must be awarded through a post-graduate certification process. Conversely, their coun- terparts from a Canadian university suggested the inclusion of a set of specific training courses like a laboratory data analysis course aimed at achieving a successful prescribing This study was limited by a small sample size, which could affect the generalizability of the findings. However, given that there is only one school of pharmacy in the state of Qatar with an average of 25 students per professional year, the study has reported a high response rate. Another limitation of this study was the participants’ susceptibility to social desirability bias since the principal investigators were their professors. The pharmacy students and graduates maybe prone to cog- nitive bias or Dunning–Kruger effect by over polishing the image of their college and its curriculum. As a result, we have 1 3 3 121 International Journal of Clinical Pharmacy (2020) 42:110–123 conducted a thorough curriculum mapping to better evaluate the quality and comprehensiveness of the curriculum of the CPH at QU with respect to prescribing-related content. 9. Auta A, Strickland-Hodge B, Maz J. Stakeholders’ views on grant- ing prescribing authority to pharmacists in Nigeria: a qualitative study. Int J Clin Pharm. 2016;38(4):960–7. y ( ) 10. Cope L, Abuzour A, Tully M. Nonmedical prescribing: where are we now? Ther Adv Drug Saf. 2016;7(4):165–72. Conclusion Pharmacists and prescribing rights: review of international developments. J Pharm Pharm Sci. 2005;8:217–25. 22. 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https://openalex.org/W3165431420	https://www.nature.com/articles/s41388-021-01839-w.pdf	English	null	Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in colorectal cancer	Oncogene	2,021	cc-by	10,555	Abstract Emerging evidences suggest that long noncoding RNA (lncRNA) plays a vital role in tumorigenesis and cancer progression. Here, the aim of this study is to investigate the biological function of long intervening noncoding RNA Linc00284 in colorectal cancer (CRC). The expression levels of Linc00284, miR-27a and c-Met were evaluated by qPCR and/or Western blotting. Immunohistochemistry was used to detect the expression of Ki67 and Phh3 in tumor tissues. The interaction between Linc00284, miR-27a and c-Met was validated by luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Cell function experiments, including CCK-8, wound-healing and transwell invasion assays, were conducted. The in vivo studies were performed with the subcutaneous tumor xenograft mouse models. Our ﬁndings reveal that Linc00284 is upregulated in CRC tissues and colorectal cancer cell lines HCT116 and SW480 in comparison with corresponding para- carcinoma tissues and human fetal colonic mucosa cells FHC. High expression of Linc00284 in tumor tissues is associated with tumor metastasis and predicts a poor clinical outcome in CRC patients. Serum Linc00284 is increased, while miR-27a is decreased in CRC patients compared to healthy controls. ROC curve analysis indicates that serum Linc00284 and miR- 27a produce the area under the curve (AUC) value of at 0.8151 and 0.7316 in patients with colorectal cancer compared to healthy individuals, respectively. Additionally, results in vitro and in vivo experiments suggest that Linc00284 silencing signiﬁcantly suppresses CRC cell proliferation and/or invasion. Mechanistically, Linc00284 promotes c-Met expression by acting as miR-27a sponge, leading to the activation of downstream signaling pathways, thereby causing malignant phenotypes of CRC cells. Taken together, Linc00284 exhibits oncogenic function and the disturbance of Linc00284/miR- 27a/c-Met regulatory axis contributes to CRC progression, providing new insight into the pathogenesis of colorectal cancer. Importantly, the expression levels of serum Linc00284 and miR-27a may serve as clinical biomarkers for CRC diagnosis. Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in colorectal cancer Received: 20 June 2020 / Revised: 29 April 2021 / Accepted: 13 May 2021 / Published online: 28 May 2021 © The Author(s) 2021. This article is published with open access Oncogene (2021) 40:4151–4166 https://doi.org/10.1038/s41388-021-01839-w Oncogene (2021) 40:4151–4166 https://doi.org/10.1038/s41388-021-01839-w ARTICLE ARTICLE ARTICLE Results To further determine the biological function of Linc00284 in CRC progression, xenograft nude mouse model was used in this study. Our ﬁndings demonstrated that the silencing of Linc00284 signiﬁcantly inhibited tumor growth in vivo without adverse effect on body weight of mice (Fig. 3A–C); meanwhile, the expression levels of cell proliferation markers Ki67 and Phosphohistone-H3 (pHH3) were markedly reduced in Linc00284 knockdown group in comparison to the control group (Fig. 3D, E). Notably, the expression levels of miR-27a, pro-apoptotic gene (Bax and Bid), tumor sup- pressor gene P53, epithelial markers (E-Cadherin and Cyto- keratin19) were signiﬁcantly upregulated, whereas the expression levels of oncogene c-Met, genes involved in cell cycle progression, including CDK4, CDK6 and CyclinD1, mesenchymal markers (N-Cadherin and Vimentin) and anti- apoptotic gene Bcl-2 were downregulated in tumor tissues (Linc00284 was silenced) compared to the control samples (Fig. 3F). In contrast, the overexpression of Linc00284 pro- moted CRC cell growth and produced the opposite effects on the above-mentioned gene expressions in tumor tissues (Fig. 3G–I). Together, these ﬁndings point to a tumor-promoting role for Linc00284 in CRC. Introduction Colorectal cancer (CRC) is one of the most common malignancies worldwide [1]. Although the therapeutic effect of surgical resection with adjuvant chemoradiotherapy or targeted therapy in CRC has been greatly improved during the past decades [2], the prognosis of patients with advanced CRC is still very poor [3]. Therefore, it is parti- cularly important to ﬁnd effective biomarkers and targets for the early diagnosis and treatment of CRC. These authors contributed equally: Jun You, Jiayi Li, Chunlin Ke, Yanru Xiao, Chuanhui Lu * Chunlin Ke ke_chunlin@yeah.net * Yanjun Mi Miyj77@xmu.edu.cn * Rongmu Xia xiarongmu@126.com * Qiyuan Li liqiyuanxm@126.com Extended author information available on the last page of the article Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41388- 021-01839-w. These authors contributed equally: Jun You, Jiayi Li, Chunlin Ke, Yanru Xiao, Chuanhui Lu These authors contributed equally: Jun You, Jiayi Li, Chunlin Ke, Yanru Xiao, Chuanhui Lu Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41388- 021-01839-w. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41388- 021-01839-w. * Chunlin Ke ke_chunlin@yeah.net * Yanjun Mi Miyj77@xmu.edu.cn * Rongmu Xia xiarongmu@126.com * Qiyuan Li liqiyuanxm@126.com Long noncoding RNA (lncRNA) is a class of RNA longer than 200 nucleotides and lacks a complete open reading frame (ORF), including LincRNA [4–6]. Increasing studies have shown that LincRNAs are abnormally expressed in various types of cancers, including colorectal cancer [5, 7], where LincRNA acts as a tumor suppressor or oncogene, inhibiting or promoting tumorigenesis and cancer progression. Extended author information available on the last page of the article 4152 J. You et al. colorectal cancers has been suggested to be a useful bio- marker for cancer diagnosis and prognosis. For instance, linc01503 and linc-ITGB1 promote CRC pro- gression and metastasis [8, 9]. Linc00284 is a newly discovered LincRNA. Previous work has showed that Linc00284 is implicated in angiogenesis during ovarian can- cer development by recruiting NF-κB1 and down-regulating mesoderm-speciﬁc transcript (MEST) [10]. Furthermore, new evidence suggested that Linc00284 was involved in the pro- gression of gastric cancer [11], primary papillary thyroid carcinoma [12], breast cancer [13], and hepatocellular carci- noma [14]. However, the underlying molecular mechanism of Linc00284 in CRC remains unclear. Linc00284 knockdown represses CRC cell proliferation and growth in vitro and in vivo To understand the role of Linc00284 in CRC cells, the expression of Linc00284 in normal colonic epithelial cells FHC and CRC cells HCT116 and SW480 was detected by qPCR analysis. Results showed that Linc00284 expression in HCT116 and SW480 cells was much higher than that in FHC cells (Fig. 2A). Further, Linc00284-knockdown HCT116 and SW480 cell lines were established and qPCR analysis con- ﬁrmed that Linc00284 expression was signiﬁcantly down- regulated in two types of cells in comparison to the control cells (Fig. 2B). After extraction of the nucleus and cytoplasm, we found that Linc00284 was mainly localized in the cyto- plasm of FHC, HCT116 and SW480 cells (Fig. 2C). CCK-8 assay showed that there was no signiﬁcant difference in cell viability of sh-Control cells and wild-type (WT) cancer cells; however, the cell viability of Linc00284-knockdown CRC cells signiﬁcantly reduced compared to the control cells (Fig. 2D). Moreover, colony formation assay and Ki-67 staining conﬁrmed that the proliferative capability of Linc00284-knockdown CRC cells was signiﬁcantly sup- pressed compared to the control cells (Fig. 2E–G). Addition- ally, wound healing and transwell invasion assay showed that Linc00284 knockdown strikingly attenuated the migratory and invasive abilities of HCT116 and SW480 cells (Fig. 2H, I). Conversely, Linc00284 overexpression promoted the malig- nant phenotypes of CRC cells (Supplementary Fig. S1). LncRNA-miRNA-mRNA network plays an important regulatory role in the pathogenesis of CRC [15], of which microRNAs (miRNAs) are endogenous noncoding RNAs with approximately 22 nucleotides and can bind to the 3′ untranslated region (3′UTR) of target messenger RNA (mRNA), regulating gene expression [16, 17]. miRNAs are broadly involved in various pathological processes, including tumor initiation and progression [18–20]. Recent work in CRC has suggested that miR-27a facilitates color- ectal cancer progression via activation of Wnt/β-catenin pathways [21, 22]. Additionally, it is worth noting that lincRNA, including Linc00284, regulates gene expression by competitively binding endogenous miRNA [5, 7]. In present study, we aim to investigate the biological functions of Linc00284 in colorectal cancer. Our ﬁndings demon- strated that targeting Linc00284 holds considerable promise for clinical anticancer therapy. Linc00284 is overexpressed in CRC and high Linc00284 expression predicts poor outcome in patients with colorectal cancer In this study, qPCR analysis revealed that the expression of Linc00284 was signiﬁcantly upregulated in colorectal can- cer tissues compared with adjacent normal tissues (N = 73, Fig. 1A, B). Identical results were obtained by immuno- ﬂuorescence (Fig. 1C). Besides, Linc00284 overexpression was associated with cancer metastasis (Fig. 1D), relapse (Fig. 1E), TNM staging (Fig. 1F) and poor overall survival (Fig. 1G). Additionally, Linc00284 level was increased in the serum of patients with colorectal cancer and sig- niﬁcantly associated with TNM stage, tumor recurrence and distant metastasis (Fig. 1H). ROC curve analysis indicated that the area under the ROC curve (AUC) value of Linc00284 level reached at 0.8151 in the peripheral blood serum of patients with colorectal cancer (n = 73), as com- pared with the healthy controls (n = 43). These data sug- gested that Linc00284 may play an important role in CRC progression and serum Linc00284 level in patients with Fig. 1 Linc00284 overexpression is associated with the clin- icopathological characteristics and poor prognosis in patients with colorectal cancer. A, B Linc00284 expression in 73 human CRC samples and paired adjacent normal tissues. C The expression of Linc00284 in CRC and adjacent normal tissues was detected by immunoﬂuorescence. Scale bar, 100 μm. D–F The expression level of Linc00284 was signiﬁcantly higher in tumor tissues of (D) patients with metastatic CRC than those without metastasis or (E) patients with recurrent CRC compared with those without recurrent CRC or (F) patients with stage I-II CRC than those with stage III-IV CRC. G Kaplan–Meier curve of overall survival. H The level of serum Linc00284 in patients with CRC and healthy controls was measured by qPCR analysis. Data are presented as the mean ± S.D. from three independent experiments. P values were calculated using two-sided paired t-test. P < 0.01; P < 0.001. Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4153 Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4153 recurrent CRC compared with those without recurrent CRC or (F) patients with stage I-II CRC than those with stage III-IV CRC. G Kaplan–Meier curve of overall survival. H The level of serum Linc00284 in patients with CRC and healthy controls was measured by qPCR analysis. Data are presented as the mean ± S.D. from three independent experiments. P values were calculated using two-sided paired t-test. Linc00284 is overexpressed in CRC and high Linc00284 expression predicts poor outcome in patients with colorectal cancer P < 0.01; P < 0.001. Fig. 1 Linc00284 overexpression is associated with the clin- icopathological characteristics and poor prognosis in patients with colorectal cancer. A, B Linc00284 expression in 73 human CRC samples and paired adjacent normal tissues. C The expression of Linc00284 in CRC and adjacent normal tissues was detected by immunoﬂuorescence. Scale bar, 100 μm. D–F The expression level of Linc00284 was signiﬁcantly higher in tumor tissues of (D) patients with metastatic CRC than those without metastasis or (E) patients with Fig. 2 Linc00284 knockdown represses CRC cell proliferation, migration and invasion in vitro. A Linc00284 expression in normal colon epithelial cells FHC, HCT116 and SW480 cells was determined by qPCR analysis. B Linc00284 silencing efﬁciency of HCT116 and SW480 cells was veriﬁed by qPCR analysis. C The levels of nuclear Linc00284 and cytosolic Linc00284 were evaluated by qPCR analysis after separating the nucleoplasm from the cytoplasm. D The cell via- bility of Linc00284-silenced CRC cells and the control cells was measured using Cell Counting Kit-8 (CCK-8) assay. E Colony for- mation assays was performed to test cell proliferative ability. F Pro- liferation of cancer cells was analyzed with Ki67 staining by ﬂow cytometry. G Quantiﬁcation of Ki67 staining by ﬂow cytometry. H The migration of Linc00284-silenced CRC cells was detected by wound healing assay. I The invasion of Linc00284-silenced CRC cells was analyzed by transwell assay. Data are presented as the mean ± S.D. from three independent experiments. Comparisons between two groups were analyzed using two-sided unpaired t-test. Multiple com- parisons were analyzed using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.01; *P < 0.001. sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. 4154 J. You et al. J. You et al. 4154 cytometry. G Quantiﬁcation of Ki67 staining by ﬂow cytometry. H The migration of Linc00284-silenced CRC cells was detected by wound healing assay. I The invasion of Linc00284-silenced CRC cells was analyzed by transwell assay. Data are presented as the mean ± S.D. from three independent experiments. Comparisons between two groups were analyzed using two-sided unpaired t-test. Multiple com- parisons were analyzed using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.01; *P < 0.001. sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. Fig. 2 Linc00284 knockdown represses CRC cell proliferation, migration and invasion in vitro. Linc00284 is overexpressed in CRC and high Linc00284 expression predicts poor outcome in patients with colorectal cancer A Linc00284 expression in normal colon epithelial cells FHC, HCT116 and SW480 cells was determined by qPCR analysis. B Linc00284 silencing efﬁciency of HCT116 and SW480 cells was veriﬁed by qPCR analysis. C The levels of nuclear Linc00284 and cytosolic Linc00284 were evaluated by qPCR analysis after separating the nucleoplasm from the cytoplasm. D The cell via- bility of Linc00284-silenced CRC cells and the control cells was measured using Cell Counting Kit-8 (CCK-8) assay. E Colony for- mation assays was performed to test cell proliferative ability. F Pro- liferation of cancer cells was analyzed with Ki67 staining by ﬂow Linc00284 binds directly to miR-27a and negatively regulates the expression of miR-27a online bioinformatics software (Starbase v3.0) was applied. It was predicted the binding sites between Linc00284 and miR- 27a (Fig. 4A). Dual-luciferase reporter assay revealed that miR-27a mimics decreased the luciferase activity of the WT- Linc00284 reporters, but not that of Mutant-Linc00284 in 293T cells, suggesting that Linc00284 directly bound to LincRNAs act as competing endogenous RNA (ceRNA) of microRNA, thereby regulating the expression of cancer- related genes. To determine the target miRNA of Linc00284, Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4155 Fig. 3 Linc00284 knockdown inhibits CRC cell growth in vivo. A Tumor diameter in the Linc00284-silenced group and the Control group (N = 6 mice per group). B Quantitative of xenograft tumor volume. C Body weight of all mice. D, E Immunohistochemistry was conducted to detect the expression levels of Ki-67 and pHH3 in tumor tissues and adjacent normal samples. F The mRNA expression of Linc00284, miR-27a, c-Met, Bax, Bcl-2, CyclinD1, CDK4, CDK6, P53, E-Cadherin, N-Cadherin, Vimentin and Cytokeratin-19 in tumor tissues with silencing of Linc00284 and control samples. G Tumor diameter in the Linc00284-overecpressed group and the control group (N = 6 mice per group). H Tumor volume in xenograft mouse model with/without overexpression of Linc00284. I The mRNA expression of Linc00284, miR-27a, c-Met, Bax, Bcl-2, CyclinD1, CDK4, CDK6, P53, E-Cadherin, N-Cadherin, Vimentin and Cytokeratin-19 in tumor tissues with overexpression of Linc00284 and the control samples. Data are presented as the mean ± S.D. from three independent experiments. P values were calculated using two-sided unpaired t-test. P < 0.01; *P < 0.001. E-cad E-Cadherin, N-Cad N-Cad- herin, CK-19 Cytokeratin-19, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. (N = 6 mice per group). H Tumor volume in xenograft mouse model with/without overexpression of Linc00284. I The mRNA expression of Linc00284, miR-27a, c-Met, Bax, Bcl-2, CyclinD1, CDK4, CDK6, P53, E-Cadherin, N-Cadherin, Vimentin and Cytokeratin-19 in tumor tissues with overexpression of Linc00284 and the control samples. Data are presented as the mean ± S.D. from three independent experiments. P values were calculated using two-sided unpaired t-test. P < 0.01; *P < 0.001. E-cad E-Cadherin, N-Cad N-Cad- herin, CK-19 Cytokeratin-19, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. (N = 6 mice per group). H Tumor volume in xenograft mouse model with/without overexpression of Linc00284. I The mRNA expression of Linc00284, miR-27a, c-Met, Bax, Bcl-2, CyclinD1, CDK4, CDK6, P53, E-Cadherin, N-Cadherin, Vimentin and Cytokeratin-19 in tumor tissues with overexpression of Linc00284 and the control samples. Data are presented as the mean ± S.D. from three independent experiments. P values were calculated using two-sided unpaired t-test. P < 0.01; **P < 0.001. E-cad E-Cadherin, N-Cad N-Cad- herin, CK-19 Cytokeratin-19, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. Fig. 3 Linc00284 knockdown inhibits CRC cell growth in vivo. A Tumor diameter in the Linc00284-silenced group and the Control group (N = 6 mice per group). B Quantitative of xenograft tumor volume. C Body weight of all mice. D, E Immunohistochemistry was conducted to detect the expression levels of Ki-67 and pHH3 in tumor tissues and adjacent normal samples. F The mRNA expression of Linc00284, miR-27a, c-Met, Bax, Bcl-2, CyclinD1, CDK4, CDK6, P53, E-Cadherin, N-Cadherin, Vimentin and Cytokeratin-19 in tumor tissues with silencing of Linc00284 and control samples. G Tumor diameter in the Linc00284-overecpressed group and the control group 4156 J. You et al. miR-27a (Fig. 4B). Consistently, RIP analysis determined that both Linc00284 and miR-27a were enriched on anti- To further unravel the biological functions of Linc00284 and miR-27a, HCT116 and SW480 cells were transfected Fig. 4 Linc00284 directly binds to miR-27a and negatively reg- ulates miR-27a expression. A Schematic diagram of the potential binding sites between Linc00284 and miR-27a. B Dual-luciferase reporter assay and C RIP experiment conﬁrmed the interaction between Linc00284 and miR-27a. D Binding of miR-27a and Ago2 determined by RIP assay. E The expression level of miR-27a in Linc00284 knockdown CRC cells. F miR-27a level in HCT116 cells transfected with pcDNA Linc00284 vectors or si Linc00284 for 48 h. G miR-27a expression in SW480 cells transfected with pcDNA Linc00284 or si Linc00284 for 48 h. H miR-27a expression in human CRC tissues was detected by qPCR analysis (N = 73). I Pearson correlation test suggested a negative correlation between Linc00284 and miR-27a expression. J Serum miR-27a level in patients with CRC (N = 73) compared to healthy controls (N = 43). Data are presented as the mean ± S.D. from three independent experiments. Comparisons between two groups were analyzed using two-sided paired t-test. Multiple comparisons were analyzed using one-way analysis of var- iance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.05; P < 0.01; P < 0.001; NS not signiﬁcant difference, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. Fig. 4 Linc00284 directly binds to miR-27a and negatively reg- ulates miR-27a expression. A Schematic diagram of the potential binding sites between Linc00284 and miR-27a. B Dual-luciferase reporter assay and C RIP experiment conﬁrmed the interaction between Linc00284 and miR-27a. D Binding of miR-27a and Ago2 determined by RIP assay. E The expression level of miR-27a in Linc00284 knockdown CRC cells. c-Met is a direct target of miR-27a and Linc00284 regulates c-Met expression by regulating miR-27a By searching on Target Scan [24] (http://www.targetscan. org/vert_72/), it predicted c-Met as a potential target for miR-27a (Fig. 6A). Dual-luciferase reporter assay validated the binding sites between miR-27a and the 3′UTR of c-Met mRNA (Fig. 6B). RIP assay further conﬁrmed the interac- tion between miR-27a and c-Met (Fig. 6C, D). Besides, the mRNA expression of c-Met gene in 73 CRC tissues was signiﬁcantly upregulated compared to the matched adjacent normal tissues (Fig. 6E). Pearson correlation analysis showed a negative correlation between the expression of c- Met and miR-27a level in CRC samples (Fig. 6F). Con- versely, c-Met expression was positively correlated with Linc00284 expression level in CRC tissues (Fig. 6G). Furthermore, c-Met expression was strikingly reduced in Linc00284 knockdown CRC cells, but miR-27a inhibitor partially reversed inhibition of c-Met expression induced by Linc00284 silencing at the transcriptional and translational levels (Fig. 6H). To the contrary, Linc00284 overexpression resulted in the up-regulation of c-Met expression, whereas miR-27a mimics treatment partially reversed c-Met expression caused by Linc00284 overexpression (Fig. 6I). Meanwhile, the expression of c-Met and its downstream molecules were monitored by qPCR analysis. Data showed that overexpression of Linc00284 in CRC cells signiﬁcantly upregulated the mRNA expression of genes involved in cell cycle progression, including CDK4, CDK6 and CyclinD1, Besides, as compared with the Linc00284 knockdown group, miR-27a inhibitor treatment was able to reverse the reduced proliferative capacity of CRC cells caused by Linc00284 knockdown (Fig. 5H, I). These in vivo studies corroborated our in vitro ﬁndings and indicated that Linc00284 promoted the malignant behaviors of CRC cells in vitro and tumor growth in vivo by targeting miR-27a. si-Linc00284, miR-27a expression was markedly elevated, whereas enforced expression of Linc00284 resulted in decreased miR-27a level in CRC cells (Fig. 4F, G). qPCR analysis further suggested that miR-27a expression was sig- niﬁcantly downregulated in human CRC tissues (Fig. 4H) and negatively correlated with Linc00284 expression (Fig. 4I). These ﬁndings indicated that Linc00284 targeted directly to miR-27a and negatively regulated miR-27a expression. miR-27a has proved to be a useful diagnostic and/or prognostic biomarker in various types of solid cancers [23]. In this study, the level of miR-27a in serum samples from patients with colorectal cancer (n = 73) was strikingly decreased as compared to the healthy individuals (n = 43, Fig. 4J). Besides, the level of miR-27a rises in serum cor- related with the TNM stage, tumor recurrence and metastasis. Given that miR-27a and miR-27b are two highly homologous microRNAs, we implemented the dual- luciferase reporter assay against miR-27b as described above, and the results conﬁrmed that the addition of exo- genous miR-27b mimics signiﬁcantly reduced the luciferase activity in WT-Linc00284 (not Mutant-Linc00284) vectors- transfected 293T cells, indicating that miR-27b was also directly bind to Linc00284 (Supplementary Fig. S4A). However, RIP assay did not yield the same results (Sup- plementary Fig. S4B-C). To explain the difference between these two experiments, we examined the expression levels of both miR-27a and miR-27b in colorectal cancer cells, and data showed that miR-27a expression was much higher than that of miR-27b, which might be the reason why it could not be detected by RIP assay (Supplementary Fig. S4D). In the subsequent cellular functional experiments, we found that exogenous miR-27b mimics in CRC cells signiﬁcantly inhibited CRC cell proliferation, migration and invasion, while miR-27b inhibitors did not have such an effect (Supplementary Fig. S4E-G). Taken together, although our ﬁndings suggest that miR-27b also binds to Linc00284, miR-27b is expressed in colorectal cancer cells at a much lower level than miR-27a. Hence, miR-27a is considered as the main downstream target of Linc00284 in this study. F miR-27a level in HCT116 cells transfected with pcDNA Linc00284 vectors or si Linc00284 for 48 h. G miR-27a expression in SW480 cells transfected with pcDNA Linc00284 or si Linc00284 for 48 h. H miR-27a expression in human CRC tissues was detected by qPCR analysis (N = 73). I Pearson correlation test suggested a negative correlation between Linc00284 and miR-27a expression. J Serum miR-27a level in patients with CRC (N = 73) compared to healthy controls (N = 43). Data are presented as the mean ± S.D. from three independent experiments. Comparisons between two groups were analyzed using two-sided paired t-test. Multiple comparisons were analyzed using one-way analysis of var- iance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.05; P < 0.01; P < 0.001; NS not signiﬁcant difference, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. To further unravel the biological functions of Linc00284 and miR-27a, HCT116 and SW480 cells were transfected with pcDNA-00284, the small interfering RNA targeting Linc00284 (si-Linc00284) or their corresponding control vectors. We found that the silencing efﬁciency of si- Linc00284 could maintain 5 days (Supplementary Fig. S3). When CRC cells were transiently silenced Linc00284 by miR-27a (Fig. 4B). Consistently, RIP analysis determined that both Linc00284 and miR-27a were enriched on anti- Ago2 magnetic beads (Fig. 4C, D). FISH assay indicated that both Linc00284 and miR-27a were expressed in the cyto- plasm of CRC cells (Supplementary Fig. S2). Moreover, Linc00284 knockdown contributed to the up-regulation of miR-27a (Fig. 4E). Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4157 and colony formation assays (Fig. 5D) revealed that the down-regulation of miR-27a rescued the inhibitory effect of linc00284 knockdown on CRC cell proliferation. Similar results were obtained in Ki-67 staining (Fig. 5E). Wound healing (Fig. 5F) and transwell invasion assays (Fig. 5G) uncovered that miR-27a inhibitor could partially reverse the inhibition of cell migratory and invasive abilities caused by Linc00284 knockdown. Further, the effect of Linc00284/ miR-27a regulatory axis on tumor growth was veriﬁed by subcutaneous tumor xenograft mouse models. Results conﬁrmed that Linc00284 knockdown could signiﬁcantly reduce cell proliferation in vivo compared to the control group. miR-27a mimics treatment in combination with Linc00284 knockdown displayed a synergistic inhibitory effect on tumor cell growth in comparison to the Linc00284 knockdown group. Linc00284 facilitates the malignant behaviors of CRC cells by targeting miR-27a To determine the effect of the Linc00284/miR-27a reg- ulatory axis on CRC cells, the proliferative and invasive capabilities of CRC cells were measured by CCK-8, cell colony formation, transwell assays. We found that miR-27a expression in CRC cells (HCT116 and SW480) was sig- niﬁcantly lower than that in normal colonic epithelial cells FHC (Fig. 5A). Next, HCT116 and SW480 cells with stably silencing Linc00284 were transfected with exogenous miR- 27a mimics or inhibitor. The transfection efﬁciency was determined by qPCR analysis (Fig. 5B). CCK-8 (Fig. 5C) mesenchymal markers (N-Cadherin and Vimentin) and anti- Cytokeratin19) were downregulated (Supplementary Fig. Fig. 5 Linc00284 suppresses the proliferation, migration and invasion of CRC cells by targeting miR-27a. A miR-27a expression in normal colonic epithelial cells FHC and CRC cell lines (HCT116 and SW480) was detected by qPCR analysis. B The effect of miR-27a inhibitor on the expression of miR-27a in Linc00284 knockdown CRC cells. Left, HCT116 cells; right, SW480 cells. C The effect of miR-27a inhibitor on CRC cell proliferation was measured by CCK-8 assay. Left, HCT116 cells; right, SW480 cells. D The effect of miR-27a inhibitor on the proliferation of Linc00284 knockdown CRC cells was determined by colony formation assay. E Ki-67 staining was used to test the effect of miR-27a inhibitor on the proliferation of Linc00284 knockdown CRC cells. F The migration ability of CRC cells was measured by wound healing assay. G The invasive ability of CRC cells was mon- itored by transwell assay. Xenograft tumors model for effect of Linc00284 knockdown alone or in combination with miR-27a (H) mimics/(I) inhibitor treatment on the growth of HCT116 xenografts. Tumor growth was monitored by measurements of tumor diameter using calipers (left); the tumor volume (middle); the expression of Linc00284, miR-27a and c-Met in tumor tissues (right). Data are pre- sented as the mean ± S.D. from three independent experiments. P values were calculated using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.05; P < 0.01; P < 0.001. sh 00284, lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. 4158 J. You et al. J. You et al. 4158 Fig. 5 Linc00284 suppresses the proliferation, migration and invasion of CRC cells by targeting miR-27a. A miR-27a expression in normal colonic epithelial cells FHC and CRC cell lines (HCT116 and SW480) was detected by qPCR analysis. Linc00284 facilitates the malignant behaviors of CRC cells by targeting miR-27a E The expression of c-Met in 73 paired CRC tissues and adjacent tissues was tested by qPCR analysis. F Pearson correlation analysis showed the correlation between c-Met and miR-27a expres- sion. G The correlation between c-Met and Linc00284 expression. H The effects of Linc00284 knockdown or in combination with miR-27a inhibitor treatment for 48 h on the mRNA and protein expression of c-Met in HCT116 cells. I The effects of Linc00284 overexpression or in combination with miR-27a mimics treatment for 48 h on the mRNA and protein expression of c-Met in HCT116 cells. Data are presented as the mean ± S.D. from three independent experi- ments. Comparison between two groups were analyzed using two-sided paired student’s t-test. Multiple comparisons were analyzed using one- way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.01; P < 0.001; NS not signiﬁcant difference, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA, OE 00284 overexpression of Linc00284. miR-27a inhibitor treatment for 48 h on the mRNA and protein expression of c-Met in HCT116 cells. I The effects of Linc00284 overexpression or in combination with miR-27a mimics treatment for 48 h on the mRNA and protein expression of c-Met in HCT116 cells. Data are presented as the mean ± S.D. from three independent experi- ments. Comparison between two groups were analyzed using two-sided paired student’s t-test. Multiple comparisons were analyzed using one- way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.01; **P < 0.001; NS not signiﬁcant difference, sh00284 lentiviral vector-mediated Linc00284 silencing by short hairpin RNA, OE 00284 overexpression of Linc00284. Fig. 6 Linc00284 regulates c-Met expression by adsorbing miR- 27a. A Schematic diagram of the binding sites between miR-27a and 3′UTR of c-Met mRNA. B The luciferase activity was tested after co- transfection with wild type (WT) 3′UTR of c-Met mRNA/mutant 3′ UTR of c-Met mRNA and miR-27a mimics for 48 h. RIP assay was used to determine the interaction between (C) miR-27a and (D) c-Met in CRC cells. E The expression of c-Met in 73 paired CRC tissues and adjacent tissues was tested by qPCR analysis. F Pearson correlation analysis showed the correlation between c-Met and miR-27a expres- sion. G The correlation between c-Met and Linc00284 expression. H The effects of Linc00284 knockdown or in combination with Linc00284 facilitates the malignant behaviors of CRC cells by targeting miR-27a B The effect of miR-27a inhibitor on the expression of miR-27a in Linc00284 knockdown CRC cells. Left, HCT116 cells; right, SW480 cells. C The effect of miR-27a inhibitor on CRC cell proliferation was measured by CCK-8 assay. Left, HCT116 cells; right, SW480 cells. D The effect of miR-27a inhibitor on the proliferation of Linc00284 knockdown CRC cells was determined by colony formation assay. E Ki-67 staining was used to test the effect of miR-27a inhibitor on the proliferation of Linc00284 knockdown CRC cells. F The migration ability of CRC cells was measured by wound healing assay. G The invasive ability of CRC cells was mon- itored by transwell assay. Xenograft tumors model for effect of Linc00284 knockdown alone or in combination with miR-27a (H) mimics/(I) inhibitor treatment on the growth of HCT116 xenografts. Tumor growth was monitored by measurements of tumor diameter using calipers (left); the tumor volume (middle); the expression of Linc00284, miR-27a and c-Met in tumor tissues (right). Data are pre- sented as the mean ± S.D. from three independent experiments. P values were calculated using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.05; P < 0.01; P < 0.001. sh 00284, lentiviral vector-mediated Linc00284 silencing by short hairpin RNA. Cytokeratin19) were downregulated (Supplementary Fig. S5) compared to the control cells; however, transfection with miR-27a mimics partially reversed the expression mesenchymal markers (N-Cadherin and Vimentin) and anti- apoptotic gene Bcl-2), while the expression levels of pro- apoptotic gene Bax, epithelial markers (E-Cadherin and mesenchymal markers (N-Cadherin and Vimentin) and anti- apoptotic gene Bcl-2), while the expression levels of pro- apoptotic gene Bax, epithelial markers (E-Cadherin and Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4159 levels of the above genes in CRC cells with Linc00284 overexpressing. In contrast to the above results, Linc00284 Linc00284/miR-27a/c-Met axis is implicated in CRC progression Fig. 6 Linc00284 regulates c-Met expression by adsorbing miR- 27a. A Schematic diagram of the binding sites between miR-27a and 3′UTR of c-Met mRNA. B The luciferase activity was tested after co- transfection with wild type (WT) 3′UTR of c-Met mRNA/mutant 3′ UTR of c-Met mRNA and miR-27a mimics for 48 h. RIP assay was used to determine the interaction between (C) miR-27a and (D) c-Met in CRC cells. Linc00284/miR-27a/c-Met axis is implicated in CRC progression levels of the above genes in CRC cells with Linc00284 overexpressing. In contrast to the above results, Linc00284 knockdown yielded the opposite results in CRC cells; fur- thermore, miR-27a inhibitor partially reversed the changes caused by Linc00284 knockdown (Supplementary Fig. S6). Overall, Linc00284 exhibits pro-tumor function in CRC by targeting miR-27a/c-Met axis. levels of the above genes in CRC cells with Linc00284 overexpressing. In contrast to the above results, Linc00284 knockdown yielded the opposite results in CRC cells; fur- thermore, miR-27a inhibitor partially reversed the changes caused by Linc00284 knockdown (Supplementary Fig. S6). Overall, Linc00284 exhibits pro-tumor function in CRC by targeting miR-27a/c-Met axis. To understand the effect of Linc00284/miR-27a/c-Met axis on CRC cell proliferation and invasion, si-Linc00284, pcDNA3.1- Linc00284 plasmid (overexpression of Linc00284; OE 00284), pcDNA3.1-c-Met plasmid (overexpression of c-Met; OE J. You et al. 4160 Fig. 7 The role of Linc00284/miR-27a/c-Met axis in CRC cells. A, B The proliferative ability of HCT116 cells were detected by col- ony formation assay. C, D The invasive ability of HCT116 cells was measured by transwell assay. E, F The proliferation of SW480 cells were detected by colony formation assay; G, H the invasion of SW480 cells was measured by transwell assay. Data are presented as the mean ± S.D. from three independent experiments. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA), post- hoc least signiﬁcant difference (LSD) test. P < 0.05; *P < 0.01. si 00284 transient transfection of small interfering RNA to knockdown Linc00284, OE 00284 overexpression of Linc00284, si c-Met transient transfection of small interfering RNA to knockdown c-Met, OE c-Met overexpression of c-Met. Fig. 7 The role of Linc00284/miR-27a/c-Met axis in CRC cells. A B Th lif ti bilit f HCT116 ll d t t d b l were analyzed using one-way analysis of variance (ANOVA), post- h l t i iﬁ t diff (LSD) t t P 0 05 *P 0 01 i were analyzed using one-way analysis of variance (ANOVA), post- hoc least signiﬁcant difference (LSD) test. P < 0.05; *P < 0.01. si 00284 transient transfection of small interfering RNA to knockdown Linc00284, OE 00284 overexpression of Linc00284, si c-Met transient transfection of small interfering RNA to knockdown c-Met, OE c-Met overexpression of c-Met. Fig. 7 The role of Linc00284/miR-27a/c-Met axis in CRC cells. A, B The proliferative ability of HCT116 cells were detected by col- ony formation assay. Linc00284/miR-27a/c-Met axis is implicated in CRC progression C, D The invasive ability of HCT116 cells was measured by transwell assay. E, F The proliferation of SW480 cells were detected by colony formation assay; G, H the invasion of SW480 cells was measured by transwell assay. Data are presented as the mean ± S.D. from three independent experiments. Multiple comparisons addition, qPCR and Western blot analysis showed that Linc00284 overexpression signiﬁcantly elevated c-Met expres- sion, while miR-27a mimics or si-c-Met treatment reversed the results caused by Linc00284 overexpression (Supplementary Fig. S7). Conversely, Linc00284 knockdown reduced c-Met expression, but miR-27a inhibitor or pcDNA3.1 c-Met treat- ment partially reversed the results induced by Linc00284 knockdown (Supplementary Fig. S7). These results suggested that Linc00284 upregulated c-Met expression by acting as miR- 27a sponge, thereby promoting CRC progression. c-Met), si-c-Met, miR-27a mimics or inhibitor was transfected into CRC cells as indicated in Fig. 7. Our data showed that Linc00284 overexpression could promote the proliferation and invasion of HCT116 cells, while treatment with miR-27a mimics or si-c-Met partially reversed the phenotypes caused by Linc00284 overexpression (Fig. 7A–D). Linc00284 silencing attenuated CRC cell proliferation and invasion, whereas miR- 27a inhibitors or pcDNA3.1-c-Met partially reversed the phe- notypes induced by Linc00284 silencing (Fig. 7A–D). It was consistent with the results from SW480 cells (Fig. 7E–H). In Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4161 Fig. 8 c-Met inhibitor PHA665752 abolishes the effect of Linc00284 on CRC cells. A, B The proliferative ability of HCT116 cells was tested by colony formation assay. C, D The invasive ability of HCT116 cells was detected by transwell assay. E, F The pro- liferation of SW480 cells was evaluated by colony formation assay. G, H The invasion of SW480 cells was measured by transwell assay. Linc00284-knockdown/overexpressing CRC cells treated with c-Met inhibitor PHA665752 (30 ng/ml) for 48 h. Data are presented as the mean ± S.D from three independent experiments. Multiple compar- isons were analyzed using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.05, **P < 0.001. si 00284 transient transfection of small interfering RNA to knockdown Linc00284, OE 00284 overexpression of Linc00284. Fig. 8 c-Met inhibitor PHA665752 abolishes the effect of Linc00284 on CRC cells. A, B The proliferative ability of HCT116 cells was tested by colony formation assay. C, D The invasive ability of HCT116 cells was detected by transwell assay. Linc00284/miR-27a/c-Met axis is implicated in CRC progression E, F The pro- liferation of SW480 cells was evaluated by colony formation assay. G, H The invasion of SW480 cells was measured by transwell assay. Linc00284-knockdown/overexpressing CRC cells treated with c-Met inhibitor PHA665752 (30 ng/ml) for 48 h. Data are presented as the mean ± S.D from three independent experiments. Multiple compar- isons were analyzed using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. P < 0.05, ***P < 0.001. si 00284 transient transfection of small interfering RNA to knockdown Linc00284, OE 00284 overexpression of Linc00284. Discussion these ﬁndings, our study demonstrated that c-Met expression was signiﬁcantly upregulated in CRC tissues in comparison to the adjacent normal tissues. And we conﬁrmed that the interaction between miR-27a and the 3′UTR of c-Met mRNA. Pearson analysis showed that there was a negative correlation between the expression of c-Met and miR-27a in CRC samples. In contrast, c-Met was positively correlated with the expression level of Linc00284 in CRC tissues. Linc00284 positively regulates c-Met expression by spong- ing miR-27a. Importantly, the co-transfection experiments proved that changes in the levels of Linc00284, miR-27a and c-Met are related to the proliferation of CRC cells, implying that Linc00284/miR-27a/c-Met axis is participated in CRC progression. However, given the complexity of tumor microenvironment, there exist other mechanisms involved in regulating the expression of miR-27a, such as methylation modiﬁcation or regulation of RNA stability [34], which deserves further investigation. It has been shown that the dysregulation of LincRNA plays an important role in the development and progression of malignant tumors including colorectal cancer [5, 7, 25]. Here, long intervening noncoding RNA Linc00284, a newly discovered LincRNA, is upregulated and promotes tumor progression in various types of malignancy such as breast tumors and hepatocellular carcinoma [12–14]. The ectopic overexpression of Linc00284 promotes the proliferation, invasion, migration and angiogenesis of ovarian cancer cells, and negatively regulates the activation of apoptosis- related pathways [10]. Consistent with these evidences, our ﬁndings indicate that Linc00284 is signiﬁcantly upregulated in CRC tissues and cell lines. High expression of Linc00284 is positively correlated with metastasis, recurrence, tumor stage and unfavorable prognosis in patients with colorectal cancer. In addition, Linc00284 knockdown can hinder the proliferative ability and migration activity of CRC cells in vitro, and inhibits tumor growth in vivo. These ﬁndings elucidate the oncogenic function of Linc00284 in CRC. [ ] g HGF/c-Met signaling plays an important role in promoting tumor angiogenesis, growth and metastasis [35, 36]. Hepa- tocyte growth factor (HGF) binds to its receptor, c-Met, activating several signaling pathways, including mitogen- activated protein kinase (MAPK)/extracellular signal- regulated kinase (ERK), PI3K/AKT, NF-κB, and STAT3/5 pathways [29]. Therefore, HGF overexpression and over- activation play a key role in tumorigenesis including CRC [29–31]. c-Met speciﬁc inhibitor has demonstrated its excit- ing antitumor activity [37, 38]. In our study, the over- expression of Linc00284 was positively correlated with the expression level of HGF/c-Met signaling-related molecules, while Linc00284 knockdown inhibited the activation of HGF/c-Met signaling. Discussion In addition, Linc00284 overexpression can reverse the inhibitory effect of PHA665752 on CRC cells. After treatment CRC cells with recombinant HGF in combination with Lin00284 knockdown or Linc00284 overexpression, we found that Linc00284 level affected the responsiveness of CRC cells to HGF. Overall, our results conﬁrm that Linc00284 is involved in the progression of CRC by controlling HGF/c-Met signaling. Naturally, further studies will be required to fully elucidate the biological mechanisms underlying these associations in CRC. Previously, bioinformatics analysis indicated that Linc00284 was a potential target for therapeutic intervention in gastric cancer [11]. Work in primary papillary thyroid cancer (PTC) showed that the expression level of Linc00284 has a negative correlation with the overall survival of PTC patients [12]. In addition, Linc00284 is implicated in angio- genesis of ovarian cancer by recruiting NF-κB1 and sup- pressing the expression of mesoderm-speciﬁc transcript (MEST) [10]. Moreover, a biofunctional analysis reveals that the Linc00284-related ceRNA network is related to serous ovarian carcinoma (SOC) carcinogenesis [26]. In present study, Linc00284 was greatly upregulated in colorectal cancer tissues compared with adjacent normal tissues. Linc00284 overexpression was signiﬁcantly associated with clin- icopathological features, including cancer metastasis, relapse, TNM staging, and poor overall survival of CRC patients. Further, bioinformatics predicted that the binding sites between Linc00284 and miR-27a that has been proved to have inhibitory effects in a variety of human cancers, including gastric cancer [27], lung cancer [28], and CRC [21]. Meanwhile, luciferase reporter and RIP analysis veriﬁed the endogenous interaction between Linc00284 and miR-27a. A negative correlation between the expression of Linc00284 and miR-27a was observed. Linc00284 acting as miR-27a sponge promotes CRC cell proliferation and invasion. LncRNAs [39] and microRNAs [40] are emerging as therapeutic opportunities, and also as non-invasive diag- nostic or prognostic biomarkers. In present study, serum Linc00284 was increased whereas serum miR-27a was decreased in patients with colorectal cancer as compared with healthy donors, both of which were related to the TNM stage, tumor recurrence and metastasis of CRC. Therefore, the combined assay of serum Linc00284 and miR-27a is valuable in the diagnosis of colorectal cancer. However, an expanded sample size is needed, and the whether the changes of serum Linc00284 and miR-27a are the causes of the CRC, or the results deserve further exploration. c-Met is a hepatocyte growth factor receptor with tyrosine kinase activity [29, 30]. c-Met inhibitor PHA665752 abolishes the effect of Linc00284 on CRC cells and invasion (Fig. 8). Moreover, it was conﬁrmed by qPCR and Western blot analysis that the silencing of Linc00284 resulted in a signiﬁcant decrease in c-Met expression, and further treatment with PHA665752 had no effect on the expression of c-Met. Linc00284 overexpression facilitated the expression of c-Met, and further treatment with PHA665752 also had no effect on the expression of c-Met (Supplementary Fig. S8). Hepatocyte growth factor (HGF) and its receptor C-met play a critical role in cancer devel- opment and progression. In this study, HGF treatment sig- niﬁcantly promoted the proliferation of CRC cells and pcDNA3.1-Linc00284 plasmid (OE-Linc00284) transfec- tion further enhanced the proliferation of CRC cells; how- ever, si-Linc00284 transfection markedly reduced cell proliferative ability (Supplementary Fig. S9). Altogether, Linc00284 exerts its antitumor effects by modulating c-Met expression in CRC. To determine the effect of c-Met pharmacological inhibitor PHA665752 on the biological function of Linc00284 in CRC cells, the in vitro assays were performed. Our ﬁndings showed that overexpression of Linc00284 signiﬁcantly enhanced CRC cell proliferative and invasive abilities, but silencing of Linc00284 inhibited CRC cell proliferation and invasion (Fig. 8), which was consistent with the above- mentioned results. PHA665752, a selective small molecule inhibitor of c-Met, represses cell proliferation and invasion (Fig. 8). Further results demonstrated that PHA665752 could effectively counteract the effect of Linc00284 over- expression on cell proliferation and invasion (Fig. 8). PHA665752 treatment in combination with Linc00284 knockdown synergistically inhibited CRC cell proliferation J. You et al. 4162 RNA extraction and qPCR Trizol reagent was used to extract total RNA according to the instructions (Invitrogen, Thermo Fisher Scientiﬁc Inc.). cDNA was synthesized with TaqMan® Reverse Transcrip- tion Reagents (CatLog No. N8080234, Thermo Fisher Scientiﬁc Inc.). The qPCR reaction was carried out with SuperScript™IV One-Step RT-PCR System (CatLog No. 12594100, Thermo Fisher Scientiﬁc) following manu- facturer’s guidance. Gene expression was normalized to GAPDH mRNA expression. Additional Table 1 listed all primers involved in this study. Each experiment was carried out in triplicate and three independent experiments. Tissue specimens In this study, 73 CRC tissues, paired adjacent non-tumor samples, the peripheral blood of CRC patients (N = 73) and healthy donors (N = 43; male, 26; female, 17; age 59–78 years; the median age, 65) were obtained from the First Afﬁliated Hospital of Xiamen University from February 2014 to August 2019. The clinicopathological data of CRC patients are listed in Table 1. All patients haven’t received radiation or chemotherapy before surgery. Tissue samples were frozen immediately in liquid nitrogen after resection until total RNA was extracted. Serum from peripheral blood was isolated and stored at −80 °C for use. Tissues and blood samples were collected with the patient’s and donor’s informed consent. This study was approved by the Medical Ethics Committee of the First Afﬁliated Hospital of Fujian Medical University. control, miR-27a antagomir and its negative control were purchased from Genechem (Shanghai Genechem Co., Ltd., China). The small hairpin RNA of linc00284 (sh 00284) was cloned into pLVX vectors (Shanghai Genechem Co., Ltd., China) following co-transfection with the packaging plasmids (pMDLg/pRRE, pVSVG and pRSV-REV; Shanghai Genechem Co., Ltd.) into 293T cells to construct lentiviral particles. Seventy-two hours post infection, the cells were selected with 2 μg/ml puromycin for 72 h. Transfection efﬁciency was evaluated by qPCR after 72 h. Herein, the lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term Linc00284 silencing in CRC cells. In addition, pcDNA3.1- 00284 plasmid (00284), pcDNA3.1-c-Met plasmid (c-Met) and empty plasmid (vector) were constructed. The cells were transfected using lipofectamine 2000 transfection reagent (Thermo Fisher Scientiﬁc Inc.) according to the manufacturer’s instructions. Cell culture CRC cell lines (HCT116 and SW480 cells), human fetal colonic mucosa cells FHC and 293T cells used in this study were purchased from the American Type Culture Collection (Rockville, MD, USA). The cell lines were authenticated by STR proﬁling and were mycoplasma free. CRC cells and 293T cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientiﬁc Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco) and 100 U/ml penicillin/streptomycin (Gibco). FHC cells were maintained in DMEM/F12 medium (Gibco) with cholera toxin (10 ng/ml), Hepes (10 mM), insulin (10 mM), transferrin (10 mM), hydrocortisone (100 ng/ml), 10% FBS and penicillin/streptomycin (100 U/ml). Cell cul- ture conditions were as below: 37 °C, 95% humidity and 5% carbon dioxide. Discussion Previous studies have shown that c- Met as an oncogene is upregulated in a variety of cancers, and promotes cancer initiation and progression [31, 32]. c- Met is activated by many molecules, including HGF that initiates transcription of a series of gene expressions, leading to tumor cell growth and invasion [30, 33]. Consistent with Oncogenic long intervening noncoding RNA Linc00284 promotes c-Met expression by sponging miR-27a in. . . 4163 Table 1 The clinicopathological factors of CRC patients (73 cases). Characteristics Number of cases (%) Age (year) ≤60 26 (35.62) >60 47 (64.38) Gender Male 38 (52.05) Female 35 (47.95) Tumor-Node-Metastasis stage I + II 51 (69.86) III 22 (30.14) Distant metastasis M0 43 (58.90) M1 + M2 30 (41.10) Recurrence Yes 49 (67.12) No 24 (32.88) In summary, our ﬁndings indicate that Linc00284 acts as miR-27a sponge, thereby up-regulating c-Met expression. LincRNA-associated ceRNA network plays a critical tumor-promoting role in CRC progression via regulation of HGF/c-Met signaling. Targeting Linc00284 may be a potential therapeutic target for CRC. Besides, in peripheral blood, serum levels of Linc00284 and miR-27a may be used as potential diagnostic biomarkers for colorectal cancer. Cell proliferation and colony-formation assays The cells were plated in 96-well plates. Cell proliferative capability was determined using CCK-8 kit (Beyotime Biotechnology Inc., Shanghai, China) according to the manufacturer’s instructions. Rabbit Anti-Ki67 antibody (Abcam, ab92742, with 1:150 working dilution) was used to assess cell proliferation by ﬂow cytometry. For colony formation assay, the transfected cells were cultured in 6- well plates at 1000 cells per well for 2 weeks. Then, the cells were ﬁxed, stained with 0.1% (W/V) crystal violet, and then photographed under microscope (Olympus, Japan). Formalin-ﬁxed and parafﬁn-embedded tissue specimens were prepared for immunohistochemistry. Immunohis- tochemistry (IHC) staining was performed as described previously [41]. The antibodies used in this study were showed as follows: rabbit Anti-Ki67 antibody (Abcam, ab92742, with 1:500 working dilution), mouse Anti-PHH3 Antibody (Proteintech Group, Inc., CatLog No. 66863-1-Ig, with 1:1000 working dilution). RNA immunoprecipitation (RIP) assay Protein-RNA interactions were identiﬁed using RNA immunoprecipitation analysis. Pierce™Magnetic RNA- Protein Pull-Down Kit (Thermo Fisher Scientiﬁc) was used for RIP analysis. The cells were collected and completely lysed in RIP lysis buffer following the manufacturer’s instructions. Next, RIP Lysate was incubated with human anti-Argonaute2 (Ago2) antibody magnetic beads (Milli- pore, Sigma-Aldrich LLC., St. Louis, MO, USA) or anti- mouse IgG negative control magnetic beads (Millipore). RNA bound with magnetic beads was extracted and veriﬁed by RT-qPCR. Plasmid construction and cell transfection The full-length of linc00284 sequence, the small hairpin RNA of linc00284 (sh 00284) and the small interfering RNA of c-Met (si c-Met), miR-27a mimic and its negative 4164 J. You et al. Subcutaneous xenograft mouse model All animal experiments were conducted in compliance with ethical regulations and approved by the Animal Ethics Committee of the First Afﬁliated Hospital of Xiamen Uni- versity. A total of 54 male BALB/c nude mice (6 weeks old, 18–20 g) were purchased from Model Animal Research Center of Nanjing University (Jiangsu, China) and ran- domly divided into nine groups (N = 6 per group). No blinding of animal experiments was done. Linc00284 knockdown/overexpressing HCT116 cells and the control cells (1 × 106 cells in 100-μl cell suspension per group) were implanted subcutaneously into the ﬂanks of immunodeﬁ- cient mice (6 mice/group), respectively. Additionally, seven days post-transplantation, miR-27a mimics/inhibitor treat- ments (10 μg in 50 μl of PBS) were performed intratumo- rally (i.t.) every three days until the termination of the experiment. Control mice were administrated with the same volume of PBS. Tumor growth was measured every week. On day 35, or on day 42, or on day 56 after tumor implantation, the mice were anesthetized. Tumor tissues completely resected and harvested for immunohistochem- ical (IHC) staining or qPCR analysis. According to manufacturer’s instructions, 293T cells were co-transfected with pcDNA3.1 linc00284-wt, pcDNA3.1 linc00284-mut, pcDNA3.1 c-Met-wt, pcDNA3.1 c-Met- mut, miR-27a mimic or control plasmid. After 48 h, total proteins were extracted and luciferase activity was mea- sured using Dual-Luciferase Reporter Assay System (Pro- mega, Madison, Wisconsin, USA). Statistical analysis 1. Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin. 2019;69:7–34. 2. Song M, Garrett WS, Chan AT. Nutrients, foods, and colorectal cancer prevention. Gastroenterology. 2015;148:1244–60.e1216. Data were analyzed using SPSS 22.0 statistical software SPSS (Inc., Chicago, IL, USA). Comparisons between two groups were analyzed by two-sided unpaired t-test. Multiple comparisons were analyzed using one-way analysis of variance (ANOVA), post-hoc least signiﬁcant difference (LSD) test. Differences in Linc00284, miR-27a and c-Met expression between CRC tissue samples and adjacent nor- mal tissues were analyzed using a two-sided paired t-test. Chi-square test was used to analyze the clinicopathological characteristics of CRC patients. The overall survival (OS) of patients with colorectal cancer was assessed by Kaplan–Meier curves and the log rank test. Pearson’s cor- relation analysis was applied to identify the correlation between gene expressions. Receiver operating characteristic (ROC) curves was applied to determine cut-off values to optimize the sensitivity and speciﬁcity of Linc00284 and miR-27a [42]. All statistical tests justiﬁed as appropriate and the data met the normal distribution. A P value less than 0.05 was considered as statistically signiﬁcant. All data were represented as mean ± standard deviation (SD). 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Protein sample (30 μg per lane) was loaded onto a 10% gel for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the proteins were transferred to polyvinylidene ﬂuoride (PVDF) membranes (Millipore, Burlington, MA, USA), followed by blocking with 5% BSA dissolved in tris buffered saline with tween (TBST, 0.1% [v/v] Tween-20) for 2 h at room temperature. The membranes were then immunoblotted with anti-c-Met antibody (Cell Signaling Technology, Inc., #24294, with 1:1000 dilutions) or anti-β-actin antibody (Cell Signaling Technology, Inc., #4970, with 1:1000 dilutions) overnight at 4 °C. After washing with TBST, the membranes were For analysis of cell migration, the cells (5 × 106) were planted into six-well dishes. When the cells reached 90% of conﬂuence, scratched in the cell monolayer using 200 μL sterile tip (1 mm wide). Wound healing rate was monitored using the microscope every 24 h. 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J Vet Cardiol. 2009;11:S33–40. 10 National Institute of Data Science in Health and Medicine, School of Medicine, Xiamen University, Xiamen, Fujian, PR China Jun You1,2 ●Jiayi Li3 ●Chunlin Ke 4 ●Yanru Xiao5 ●Chuanhui Lu6 ●Fakun Huang7 ●Yanjun Mi 8 ●Rongmu Xia 9 ● Qiyuan Li 10 1 Department of Gastrointestinal Oncology Surgery, Cancer Center, The First Afﬁliated Hospital of Xiamen University, Xiamen, Fujian, PR China 1 Department of Gastrointestinal Oncology Surgery, Cancer Center, The First Afﬁliated Hospital of Xiamen University, Xiamen, Fujian, PR China 6 Department of Colorectal Cancer Surgery, Xiamen Cancer Center, The First Afﬁliated Hospital of Xiamen University, Teaching Hospital of Fujian Medical University, Xiamen, Fujian, PR China 6 Department of Colorectal Cancer Surgery, Xiamen Cancer Center, The First Afﬁliated Hospital of Xiamen University, Teaching Hospital of Fujian Medical University, Xiamen, Fujian, PR China 1 Department of Gastrointestinal Oncology Surgery, Cancer Center, The First Afﬁliated Hospital of Xiamen University, Xiamen, Fujian, PR China 2 The School of Clinical Medical, Fujian Medical University, Fuzhou, Fujian, PR China 3 Department of Medical Oncology, Xiamen Key Laboratory of Antitumor Drug Transformation Research, The First Afﬁliated Hospital of Xiamen University, School of Clinical Medicine, Fujian Medical University, Xiamen, Fujian, PR China 4 Department of Radiotherapy, The First Afﬁliated Hospital of Fujian Medical University, Fuzhou, Fujian, PR China 5 Department of Medical Oncology, The First Afﬁliated Hospital of Xiamen University, Xiamen, Fujian, PR China 6 Department of Colorectal Cancer Surgery, Xiamen Cancer Center, The First Afﬁliated Hospital of Xiamen University, Teaching Hospital of Fujian Medical University, Xiamen, Fujian, PR China 2 The School of Clinical Medical, Fujian Medical University, Fuzhou, Fujian, PR China 2 The School of Clinical Medical, Fujian Medical University, Fuzhou, Fujian, PR China 7 Department of Gastrointestinal Surgery, The First Afﬁliated Hospital of Fujian Medical University, Fuzhou, Fujian, PR China 7 Department of Gastrointestinal Surgery, The First Afﬁliated Hospital of Fujian Medical University, Fuzhou, Fujian, PR China 8 Department of Medical Oncology, Xiamen Key Laboratory of Thoracic Tumor Diagnosis and Treatment, Institute of Lung Cancer, The First Afﬁliated Hospital of Xiamen University, School of Clinical Medicine, Fujian Medical University, Xiamen, Fujian, PR China 3 Department of Medical Oncology, Xiamen Key Laboratory of Antitumor Drug Transformation Research, The First Afﬁliated Hospital of Xiamen University, School of Clinical Medicine, Fujian Medical University, Xiamen, Fujian, PR China 4 Department of Radiotherapy, The First Afﬁliated Hospital of Fujian Medical University, Fuzhou, Fujian, PR China 4 Department of Radiotherapy, The First Afﬁliated Hospital of Fujian Medical University, Fuzhou, Fujian, PR China 9 School of Medicine, Xiamen University, Xiamen, Fujian, PR China 9 School of Medicine, Xiamen University, Xiamen, Fujian, PR China 5 Department of Medical Oncology, The First Afﬁliated Hospital of Xiamen University, Xiamen, Fujian, PR China 10 National Institute of Data Science in Health and Medicine, School of Medicine, Xiamen University, Xiamen, Fujian, PR China
https://openalex.org/W2098154926	https://iris.unito.it/bitstream/2318/1578029/1/art%253A10.1007%252FJHEP09%25282015%2529201.pdf	English	null	Search for neutral color-octet weak-triplet scalar particles in proton-proton collisions at s = 8 $$ \sqrt{s}=8 $$ TeV	The Journal of high energy physics/The journal of high energy physics	2,015	cc-by	18,817	Published for SISSA by Springer Received: May 29, 2015 Accepted: August 25, 2015 Published: September 29, 2015 Received: May 29, 2015 Accepted: August 25, 2015 Published: September 29, 2015 Search for neutral color-octet weak-triplet scalar particles in proton-proton collisions at √s = 8 TeV JHEP09(2015)201 Open Access, Copyright CERN, for the beneﬁt of the CMS Collaboration. The CMS collaboration E-mail: cms-publication-committee-chair@cern.ch Abstract: A search for pair production of neutral color-octet weak-triplet scalar particles (Θ0) is performed in processes where one Θ0 decays to a pair of b quark jets and the other to a Z boson plus a jet, with the Z boson decaying to a pair of electrons or muons. The search is performed with data collected by the CMS experiment at the CERN LHC corresponding to an integrated luminosity of 19.7 fb−1 of proton-proton collisions at √s = 8 TeV. The number of observed events is found to be in agreement with the standard model predictions. The 95% conﬁdence level upper limit on the product of the cross section and branching fraction is obtained as a function of the Θ0 mass. The 95% conﬁdence level lower bounds on the Θ0 mass are found to be 623 and 426 GeV, for two diﬀerent octo-triplet theoretical scenarios. These are the ﬁrst direct experimental bounds on particles predicted by the octo-triplet model. Keywords: Exotics, Hadron-Hadron Scattering Keywords: Exotics, Hadron-Hadron Scattering ArXiv ePrint: 1505.08118 ArXiv ePrint: 1505.08118 doi:10.1007/JHEP09(2015)201 doi:10.1007/JHEP09(2015)201 for the beneﬁt of the CMS Collaboration. Article funded by SCOAP3. Contents 1 Introduction 1 2 CMS detector 3 3 Event selection 3 4 Backgrounds 6 4.1 Z + jets 6 4.2 tt + jets and tW + jets 7 4.3 Diboson + jets 7 4.4 Multijet events 8 5 Systematic uncertainties 8 6 Results 11 7 Summary 12 The CMS collaboration 19 Contents 1 Introduction 2 CMS detector 3 Event selection 4 Backgrounds 4.1 Z + jets 4.2 tt + jets and tW + jets 4.3 Diboson + jets 4.4 Multijet events 5 Systematic uncertainties 6 Results 7 Summary The CMS collaboration Contents 1 Introduction 1 2 CMS detector 3 3 Event selection 3 4 Backgrounds 6 4.1 Z + jets 6 4.2 tt + jets and tW + jets 7 4.3 Diboson + jets 7 4.4 Multijet events 8 5 Systematic uncertainties 8 6 Results 11 7 Summary 12 The CMS collaboration 19 JHEP09(2015)201 7 Summary The CMS collaboration 19 1 Introduction In addition to the s-channel gluon process shown in the ﬁgure, the gluon-gluon interaction can also proceed directly through gg →Θ0Θ0 or a t-channel Θ0 exchange. The decays of the Θ0 and Z boson are also included, where the Θ0 decays are described in the main text. JHEP09(2015)201 quarks, where φI is a color singlet from a general renormalizable coloron model [15]. In the present analysis we are searching for Θ0 produced in pairs. The φI mass (mφI) is a free parameter and has an indirect impact on the Θ0 pair production cross section, via the G′ width. Unlike color-octet weak-singlet particles, octo-triplet particles do not decay to gluons. However, with a Θ0 in the loop, one-loop decays to an electroweak boson and a gluon are allowed. There is a potentially similar rate of decays to standard model (SM) quark pairs via mixing through a vector-like quark, which can be as heavy as a few TeV. These heavy vector-like quarks appear in many extensions of the SM, such as composite Higgs models [18] and little Higgs models [19]. In the former, the Higgs boson may be a bound state of a top quark and the heavy vector-like quark, where the binding is provided by the spin-1 G′ coloron. In this paper we assume that one Θ0 decays to two b quark jets and the other to a Z boson plus a jet originating from a gluon, with Z →ℓℓ, where ℓis an electron or muon. With no existing constraints on the ratio of Θ0 decays to quarks versus those decaying to Zg, we have studied a ﬁnal state that includes both decay modes. Requiring Z →ℓℓprovides a strong experimental signature for event selection and background suppression. The leading order (LO) Feynman diagrams for Θ0 pair production by quark-antiquark annihilation and gluon gluon interaction and the decays explored in this analysis are shown in ﬁgure 1 gluon-gluon interaction, and the decays explored in this analysis, are shown in ﬁgure 1. The signal production cross section for the octo-triplet model [12] is evaluated at LO with the MadGraph v4.5.1 program [20]. Figure 2 shows the values of the cross section in the 2-dimensional plane of G′ mass (mG′) and Θ0 mass (mΘ0). The masses of Θ0, G′, and φI are free parameters in general, but we use mφI = 125 GeV+mΘ0/3 as a benchmark. 1 Introduction The CERN LHC is well suited to searches for new colored particles. These searches have targeted particles including vector-like quarks (color triplet spin-1/2 particles), colorons (color octet spin-1 particles), gluinos (color octet Majorana fermions), and color-octet weak- singlet scalars [1–6]. With the recent discovery of a Higgs boson [7–9], so far consistent with being a neutral scalar colorless particle [10, 11], it is also interesting to look for additional scalars, in this case with color charge, with masses close to the electroweak scale. Particles with this combination of spin, charge, and color quantum numbers have not been thoroughly sought at collider experiments. Neutral color-octet spin-0 particles (Θ0), for example, emerge in the octo-triplet model [12], which includes three particles (Θ+, Θ0, Θ−) that transform as a color octet and weak triplet under SU(3)c × SU(2)W. The Θ0 may arise from the sector responsible for breaking extended gauge symmetry associated with colorons [13–15]. Octo-triplets may also be fermion-antifermion bound states [16, 17], or elementary particles from non-minimal grand uniﬁed theory. To date, no direct experimental bound has been set for particles predicted by the octo-triplet model. Octo-triplet particles would be produced in pairs at the LHC either through quark- antiquark annihilation or gluon-gluon interactions, with the former mediated by coloron (G′) particles or by gluons (g). If a G′ is produced, it can decay to Θ0Θ0, Θ0φI, or two – 1 – q q G′ or g Θ0 Θ0 b b e−, µ− e+, µ+ g Z g g g Θ0 Θ0 b b e−, µ− e+, µ+ g Z Figure 1. Leading order Feynman diagrams for Θ0 pair production by quark-antiquark annihila- tion, through an intermediate G′ or gluon (left), and the gluon-gluon interaction (right). In addition to the s-channel gluon process shown in the ﬁgure, the gluon-gluon interaction can also proceed directly through gg →Θ0Θ0 or a t-channel Θ0 exchange. The decays of the Θ0 and Z boson are also included, where the Θ0 decays are described in the main text. g g g Θ0 Θ0 b b e−, µ− e+, µ+ g Z q q G′ or g Θ0 Θ0 b b e−, µ− e+, µ+ g Z Figure 1. Leading order Feynman diagrams for Θ0 pair production by quark-antiquark annihila- tion, through an intermediate G′ or gluon (left), and the gluon-gluon interaction (right). 1 Introduction We explore two distinct mass scenarios: mG′ = 2.3mΘ0 and mG′ = 5mΘ0. The ﬁrst example corresponds to a scenario in which the signal cross section is enhanced because of the additional contributions from colorons (see ﬁgure 2), while the second relation corresponds to a scenario in which the contribution from colorons is negligible. For both mass relations we use Θ0 masses, ranging from 200 to 900 GeV, which are large enough to allow Θ0 decay to on-shell Z bosons, but small enough to be within the sensitivity of this search. The ﬁrst mass relation, mG′ = 2.3mΘ0, results in Θ0 pair production cross sections from 125 pb down to 2.23×10−2 pb, for the range of Θ0 masses considered. This mass relation also sets – 2 – the G′ mass suﬃciently above Θ0 pair production threshold, but small enough compared to mΘ0 that production through G′ is considerable, at 49 to 86% of the total, depending on mΘ0. In this case, quark-antiquark annihilation dominates, while gluon-gluon interaction production decreases with increasing Θ0 (and G′) mass, because of the diﬀerences in the parton distribution functions (PDF) of quarks and gluons [21]. The second mass relation considered, mG′ = 5mΘ0, results in Θ0 pair production cross sections from 63.3 pb down to 2.62×10−3 pb, for the range of Θ0 masses considered, which are a factor of two smaller than for the ﬁrst mass relation. This corresponds to a region where gluon-gluon interactions dominate and production through G′ is just 0.6 to 3.6% of the total, depending on the Θ0 mass. The cross sections discussed above agree with those calculated in ref. [22] where a similar model is considered. JHEP09(2015)201 The decay modes Θ0 →Zg and Θ0 →γg are invariant under SU(2)W. However, in the Lagrangian, the coeﬃcients of these modes turn out to be much smaller than one. This feature motivates consideration of other decay modes, which can be represented by higher- dimensional operators, and therefore are usually considered as negligible with respect to dominant decay modes. In particular, q¯q decay modes could be as large as the electroweak boson plus gluon modes [12]. In the Θ0 mass range we consider, the dominant fermion decay mode is either bb or tt depending on the values of coeﬃcients appearing in diﬀerent terms of the Θ0 →qq Lagrangian. 2 CMS detector The central feature of the CMS apparatus is a superconducting solenoid of 6 m internal diameter, providing a magnetic ﬁeld of 3.8 T. Within the solenoid volume are a silicon pixel and strip tracker, a lead tungstate crystal electromagnetic calorimeter, and a brass and scintillator hadron calorimeter, each composed of a barrel and two endcap sections. Muons are measured in gas-ionization detectors embedded in the steel ﬂux-return yoke outside the solenoid. Extensive forward calorimetry complements the coverage provided by the barrel and endcap detectors. A more detailed description of the CMS detector, together with a deﬁnition of the coordinate system used and the relevant kinematic variables, can be found in ref. [23]. 1 Introduction As a benchmark, we assign half of the branching fraction to Θ0 →bb, leaving the other half to the Θ0 →Zg and Θ0 →γg decay modes (i.e. B(Θ0 →tt) = 0). The ratio of branching fractions B(Θ0 →Zg)/B(Θ0 →γg) is set by the mass of the Θ0. For the range of Θ0 masses explored, B(Θ0 →Zg) is to 38% and B(Θ0 →γg) accounts for the remaining to 17%. The total cross section times branching fraction for the ﬁnal state studied in this analysis ranges from 2.77 pb down to 5.74×10−4 pb for the mass relation mG′ = 2.3mΘ0, and 1.40 pb down to 6.73×10−5 pb for the mass relation mG′ = 5mΘ0. 3 Event selection This analysis uses proton-proton collision data at a center-of-mass energy of 8 TeV collected with dilepton (ee, µµ, and eµ) triggers with transverse momentum pT thresholds of 17 and – 3 – mass [GeV] 0 Θ 200 300 400 500 600 700 800 900 G' mass [GeV] 1000 2000 3000 4000 5000 6000 -2 10 -1 10 1 10 2 10 [pb] σ = 8 TeV s at pair production cross section 0 Θ 0 Θ = 5 m G' m 0 Θ = 4 m G' m 0 Θ = 3 m G' m 0 Θ = 2.3 m G' m Figure 2. Cross sections for Θ0 pair production, as a function of the coloron G′ and the octo-triplet Θ0 masses. The dotted lines show a few examples of assumed linear relationship between the two masses. mass [GeV] 0 Θ 200 300 400 500 600 700 800 900 G' mass [GeV] 1000 2000 3000 4000 5000 6000 -2 10 -1 10 1 10 2 10 [pb] σ = 8 TeV s at pair production cross section 0 Θ 0 Θ = 5 m G' m 0 Θ = 4 m G' m 0 Θ = 3 m G' m 0 Θ = 2.3 m G' m JHEP09(2015)201 Figure 2. Cross sections for Θ0 pair production, as a function of the coloron G′ and the octo-triplet Θ0 masses. The dotted lines show a few examples of assumed linear relationship between the two masses. 8 GeV for the two lepton candidates. The eµ data set is used to estimate tt background. The data sample corresponds to an integrated luminosity of 19.7 fb−1. We select events with a ﬁnal state containing a Z boson and a jet (Z + jet), together with two b quark jets (b jet pair), where the Z boson decays to a pair of electrons or muons. Electrons are reconstructed using selection criteria that take into account radiated photons [24]. Candidates are required to have pT > 20 GeV and \|η\| < 2.5 where η is the pseudorapidity. Electrons from photon conversions are rejected. An isolation condition is imposed within a cone of ∆R = √ (∆η)2 + (∆φ)2 < 0.3 around the electron. 3 Event selection The open red rectangle on left indicates the signal region, as described in the text. quark, if it satisﬁes the “loose operating point” requirements of the combined secondary vertex tagger [32]. The tagging eﬃciency is about 70–90% and mistagging rate is about 10–20%, depending on pT and η of the jets. Candidate events must satisfy the following criteria: at least one reconstructed primary vertex satisfying \|z\| < 24 cm and impact parameter less than 2 cm; an opposite-sign, same- ﬂavor lepton (electron or muon) pair, sharing a primary vertex and with an invariant mass between 80 and 100 GeV to form a Z boson candidate; the two leptons separated from jets by ∆R > 0.5; at least two b-tagged jets forming a b jet pair system (sum of the b jet four-momenta); and at least one additional jet. If more than two b-tagged jets are present in the event, the jets with the largest pT values are selected for the b jet pair system. The mass of one Θ0 candidate is reconstructed from the b jet pair system, and the mass of the other Θ0 candidate is reconstructed from the combination of the Z boson candidate and the highest pT jet that is not a part of the b jet pair system. With this prescription, the correct combination of the Z boson and the jet in simulated signal samples is chosen about 65 to 80% of the time, depending on mΘ0. For each hypothetical mass point, we consider signal events in a rectangle that is formed in the two-dimensional mass plane deﬁned by the two reconstructed Θ0 masses. We determine the center, length, and width of the rectangle for each mass point by ﬁtting the b jet pair and Z+jet mass distributions in the simulated signal samples with a modiﬁed Gaussian function that has an additional parameter to account for low-mass non-Gaussian tails. The length and width of each rectangle are deﬁned as ±3σsd where σsd is the standard deviation from the modiﬁed Gaussian ﬁt. For example, the rectangle is 240 GeV long and 130 GeV wide for the signal with mG′ = 1100 GeV and mΘ0 = 478 GeV (ﬁgure 3 (left)). We choose a 3σsd wide window to keep more signal events in middle-high mass regions, since most of the SM background events appear in low-mass region (ﬁgure 3 (right)). 3 Event selection The sum of the pT of charged particles and transverse energy ET = E sin θ of neutral particles within this cone is corrected in each event for energy deposits due to additional interactions within beam bunch crossings (pileup). The corrected sum must be less than 15% of the electron pT. Muons are reconstructed using selection criteria based on quantities measured in the tracker and muon sub-detectors. Candidates are required to have pT > 20 GeV and \|η\| < 2.4. The track associated with the muon candidate is required to have hits in the pixel, silicon strip, and muon systems. An isolation condition requires that the sum of the pT of charged particles and ET of neutral particles within a cone of ∆R < 0.4 around the muon is less than 12% of the muon pT. The pileup correction for muon isolation is similar to the one applied for electrons. A particle-ﬂow technique [25, 26] is used to identify jet constituents, which are input to the FastJet algorithm [27, 28] for clustering using the anti-kT algorithm [29] with a distance parameter of 0.5. The jet energy scale (JES) is measured in data with Z/γ+jet and dijet events [30, 31], and a correction is applied to both data and simulated samples. The corrected jets must have pT > 40 GeV and \|η\| < 2.4. Jets within ∆R = 0.3 from an isolated electron or muon as deﬁned above are not counted as part of the 3 jet require- ment. Additional requirements, based on the energy balance between charged and neutral hadronic energy in the jet, are used to reduce contamination from misidentiﬁed jets. A jet is considered b tagged, consistent with originating from the hadronization of a bottom – 4 – Figure 3. Distributions of Z+jet mass versus b jet pair mass for signal events with mG′ = 1100 GeV and mΘ0 = 478 GeV (left) and background events (right), in the electron channel (distributions are similar in the muon channel). The open red rectangle on left indicates the signal region, as described in the text. JHEP09(2015)201 Figure 3. Distributions of Z+jet mass versus b jet pair mass for signal events with mG′ = 1100 GeV and mΘ0 = 478 GeV (left) and background events (right), in the electron channel (distributions are similar in the muon channel). 4.1 Z + jets More than half of the background events come from Drell-Yan (DY) production in asso- ciation with jets. There are two points in estimating the background that require special attention. First, care must be taken in estimating the yield of Z+≥3 jets events as the sim- ulation may not correctly model the kinematic properties of multijets. Second, the event yield and shape in the Z + b-jets process, where the kinematic properties of b jet pairs and the fraction of heavy-ﬂavor jets in inclusive Z + jets might also suﬀer from mis-modeling in the simulation. The following paragraphs describe how the data are used to improve on the estimates from simulations. To address the ﬁrst point, the event yield from the simulation is multiplied by a correction factor to normalize it to data in a control region. This control region is deﬁned such that the dilepton mass is between 80 and 100 GeV and there are at least three jets, none of which is b tagged. The correction factor is found to be 0.98 ± 0.12 (0.91 ± 0.12) for the electron (muon) channel. The correction is applied to all signal rectangle regions. The 12% uncertainty comes from b tagging, JES, and pileup systematic uncertainties in the control region, added in quadrature, while the statistical uncertainty is negligible. The Z + jet mass in the Z + ≥3 jets (one b tag) control region is plotted in ﬁgure 4 after the correction factor has been applied. Agreement between the data and simulated sample is observed in the Z + ≥3 jets (no b tag) and Z + ≥3 jets (one b tag) control regions. The second point requires a diﬀerent approach since control regions that include two or more b jets may suﬀer from signal contamination. We take a two-step approach. First, the simulated Z+b-jets events are weighted by the ratio of the k-factor (NLO cross section divided by LO cross section) for Z + 2 b-jets to that of Z+2 jets, using mcfm [39]. The ratios vary from 1.09 to 2.56 in the b jet pair mass range of 20 GeV to 1.8 TeV. In the simulation, about 20% of Z+jets events in the signal region have at least two jets originating from b quarks. 4 Backgrounds The dominant background processes are Z + jets and tt + jets. The contributions from other processes, including diboson+jets and multijet, are small. The samples used for all background processes are generated at tree level with the MadGraph program, inter- faced with pythia v6.4 [33] for showering and hadronization. The Z + jets background is normalized to the next-to-next-to-leading-order cross section using fewz v2.1 [34]. The tt + jets and tW+jets (single top quark) backgrounds are normalized to the next-to-next- to-leading-logarithm cross sections [35, 36]. The diboson+jets backgrounds are normalized to the next-to-leading-order (NLO) cross section from the mcfm v5.8 [37] calculation. Full simulation of the CMS detector is implemented using the Geant4 package [38]. JHEP09(2015)201 3 Event selection The signal search regions are deﬁned for the electron and muon channels separately, although these turn out to be very similar. – 5 – 4.2 tt + jets and tW + jets After accounting for the Z + jets background, most of the remaining background events come from tt + jets, with a smaller contribution from tW+jets production. Both tt + jets and tW+jets processes can yield ﬁnal states containing an opposite-sign eµ pair. In this analysis, we are considering only opposite-sign, same-ﬂavor lepton pairs, and thus the data containing an opposite-sign eµ pair can be used to calibrate the tt + jets and tW+jets backgrounds. The overall event yield in the simulation is multiplied by a factor of 1.07±0.13 to normalize it to data in the control region containing an eµ pair with the mass between 60 and 120 GeV (Zeµ) and at least three jets, at least two of which are b tagged. The uncertainty comes from b tagging, JES, and pileup systematic uncertainties in the control region, added in quadrature, while the statistical uncertainty is negligible. The b jet pair and Zeµ+jet mass distributions in this control region are plotted in ﬁgure 5 after the normalization factor is applied. 4.1 Z + jets In the second step, the remaining diﬀerence between data and simulation is evaluated as a function of b jet pair mass in the non-signal, oﬀ-diagonal regions (sidebands) in the b jet pair mass and Z + jet mass plane. The uncertainty in the Z+heavy-ﬂavor jets processes is taken from this diﬀerence or from the uncertainty in the CMS cross section measurement [40], whichever is larger. The uncertainty varies from 20 to 50%. – 6 – Figure 4. The Z + jet mass distribution in the Z + ≥3 jet (one b tag) control region after the appropriate correction factor has been applied, for the electron channel (left) and muon channel (right). The panels at the bottom show the ratio of data to background simulation, with the band representing the systematic uncertainty including the normalization uncertainty obtained from the Z + ≥3 jet (no b tag) control region. JHEP09(2015)201 Figure 4. The Z + jet mass distribution in the Z + ≥3 jet (one b tag) control region after the appropriate correction factor has been applied, for the electron channel (left) and muon channel (right). The panels at the bottom show the ratio of data to background simulation, with the band representing the systematic uncertainty including the normalization uncertainty obtained from the Z + ≥3 jet (no b tag) control region. The uncertainty in the shape of the other variable that is used to deﬁne the signal search regions, the Z + jet mass distribution, is estimated by comparing the distribution in the simulated Z+jets sample with those of several MadGraph samples that are produced with factorization or renormalization scales and matrix element parton shower matching thresholds varied up and down by a factor of two. The maximum diﬀerence between the nominal distribution and the varied distributions is taken as an uncertainty. The uncertainty varies from 2 to 55%. 4.4 Multijet events The estimation of the multijet background with simulated samples is diﬃcult given that the mis-identiﬁcation rates of jets as electrons or muons are small [24, 43]. Unlike Z →ℓℓ and W−W+ →ℓℓν¯ν processes, there are about equal amount of like-sign and opposite- sign lepton pairs in the background because the leptons are either misidentiﬁed or are non-prompt. In this analysis, we consider only opposite-sign, same-ﬂavor lepton pairs, and thus the data containing like-sign, same-ﬂavor lepton pairs can be used as a control sample to determine the size of the contribution from multijet events. After the Z boson mass window selection and the requirement of at least three jets, with at least two of them being b tagged, and after subtracting events from all other simulated background processes, 27 (13) dielectron (dimuon) events are left in the like-sign dilepton channel. This corresponds to 1.6% (0.7%) of the number of opposite-sign dilepton events. These events are distributed mostly in the lower mass region, as is the case for other backgrounds. Given the small number of like-sign events in the control sample and their mass distribution, the multijet background is found to be negligible. 4.3 Diboson + jets A few percent of the background events come from diboson+jets processes. The simulated diboson cross sections are found to be in agreement with those measured by CMS [41, 42]. Therefore we make no correction to the event yields. – 7 – Figure 5. The Zeµ+jet (left) and b jet pair mass (right) distributions in the tt control region after the appropriate correction factor is applied. The panels at the bottom show the ratio of data to background simulation, with the band representing the systematic uncertainty. JHEP09(2015)201 Figure 5. The Zeµ+jet (left) and b jet pair mass (right) distributions in the tt control region after the appropriate correction factor is applied. The panels at the bottom show the ratio of data to background simulation, with the band representing the systematic uncertainty. 5 Systematic uncertainties We organize the sources of systematic uncertainties in three categories: sources aﬀect- ing both the signal and background; sources aﬀecting only the background; and sources aﬀecting only the signal. A summary of the systematic uncertainties is given in table 1. Sources aﬀecting both the signal and background: • b tagging The data-to-simulation correction factor for b tagging is varied by its uncertainties. The resulting uncertainties in the number of predicted signal and background events range from 13 to 25%. • b tagging The data-to-simulation correction factor for b tagging is varied by its uncertainties. The resulting uncertainties in the number of predicted signal and background events range from 13 to 25%. • b tagging – 8 – • Jet energy scale The jet energies are varied by the uncertainty in the applied JES correction. The resulting event yield uncertainties range from 0.2 to 2.6% for the signal and 4 to 8% for the background. • Lepton trigger and identiﬁcation (ID) The lepton trigger and ID (including isolation) uncertainty is estimated by varying the data-to-simulation correction factor by its uncertainty, which is measured in dilepton data where at least one of the leptons passes stringent identiﬁcation criteria. The yield uncertainty is about 1%, both for the signal and the background. JHEP09(2015)201 • Pileup modeling An uncertainty in the pileup modeling in the signal and background samples is estimated by varying by 5% the total inelastic cross section, as measured at the LHC [44, 45]. The event yield uncertainty is about 1%, somewhat higher for the signal than for the background. Sources aﬀecting only the background: Sources aﬀecting only the background: • Z + jet mass shape for Z + jets background The uncertainty is evaluated by comparing the varied simulated samples as mentioned in section 4. The shape of the Z + jet mass distribution is much less certain in the high-mass tail than in the low-mass region because of the limited number of simulated events with high masses. The uncertainty in the estimated number of Z + jets events varies from 2 to 55%. • b jet pair mass shape for Z + jets background The uncertainty from this source is evaluated in the oﬀ-diagonal sidebands in the b jet pair mass and Z+jet mass plane, as mentioned in section 4, and takes into account the uncertainty in the CMS cross section measurement of Z+heavy-ﬂavor jets processes. 5 Systematic uncertainties The resultant uncertainty in the Z + jets event yield is in the range 20 to 50% • Normalization of Z + jets and tt + jets backgrounds The uncertainty in the event yield is 12% for Z + jets and 13% for tt + jets processes as mentioned in section 4. • Diboson cross section The uncertainty is taken from the underlying CMS diboson cross section measure- ments [41, 42], and implies a 10% uncertainty in the estimated number of diboson events. Sources aﬀecting only the signal: Sources aﬀecting only the signal: • Initial state radiation (ISR) modeling For the signal samples, the uncertainty from this source is estimated from the pT dis- tribution of the Θ0Θ0 system. The distribution generated using MadGraph is found to be in agreement with the data for heavy object systems with pT < 120 GeV [46]. – 9 – Source Signal [%] Z + jets [%] tt and tW [%] Diboson [%] Signal and background b tagging 13–25 15–16 13–15 16–17 Jet energy scale 0.2–2.6 6–8 4–6 5–7 Lepton ID, isolation, trigger 0.9–1.2 0.9–1.2 0.9–1.3 0.9–1.2 Pileup modeling 0.1–1.5 0.3–0.7 0.2–1.0 0.3–0.7 Background only Z + jet mass shape — 2–55 — — b jet pair mass shape — 20–50 — — Normalization — 12 13 — Diboson cross section — — — 7–10 Signal only ISR 1–5 — — — Integrated luminosity 2.6 — — — Table 1. Impact of systematic uncertainties on individual event yields. Ranges show the variation over the search regions that are considered. Dashes indicate cases where a systematic uncertainty is not applied. Sources appearing in more than one process are treated as correlated in the limit setting. JHEP09(2015)201 Table 1. Impact of systematic uncertainties on individual event yields. Ranges show the variation over the search regions that are considered. Dashes indicate cases where a systematic uncertainty is not applied. Sources appearing in more than one process are treated as correlated in the limit setting. Above 120 GeV, an uncertainty is assigned that varies from 5 to 20%, depending on the pT. This leads to a 1–5% uncertainty in the yield of signal events for the mass ranges that are considered. • Integrated luminosity The CMS experiment collected data equivalent to 19.7 fb−1 with a 2.6% uncer- tainty [47]. This uncertainty applies only to yield of signal events. 5 Systematic uncertainties The back- ground events are either normalized in a control region or the normalization is taken from data. The systematic uncertainties in event yield due to the lepton energy scale, and the lepton and jet energy resolution, are studied and found to be negligible (<0.1%) compared to the other sources of uncertainty. For the signal samples, the impact on the event yields due to PDF uncertainties are estimated by following the PDF4LHC recommendation [48, 49]. These uncertainties are not used in the limit setting, but are instead included as bands on the theoretical pre- dictions. Since the Z + jets and tt + jets backgrounds are normalized in control regions, PDF uncertainties are not applied. Similarly the diboson+jets background normalization is taken from the data [41, 42]. The uncertainty in the signal yield is 7–45%. – 10 – Figure 6. Distributions of the Z + jet mass versus b jet pair mass in data (top left and right), and estimated background (bottom left and right). The number of signal candidate events is counted in the rectangular boxes deﬁned for each signal mass hypothesis, as discussed in section 3. JHEP09(2015)201 Figure 6. Distributions of the Z + jet mass versus b jet pair mass in data (top left and right), and estimated background (bottom left and right). The number of signal candidate events is counted in the rectangular boxes deﬁned for each signal mass hypothesis, as discussed in section 3. 6 Results The numbers of observed data events in each signal region after all of the selection re- quirements are applied are consistent with the predictions from SM processes within two standard deviations, and are summarized in tables 2 and 3. Table 2 shows the number of events for signal, total background, and observed data, while table 3 shows the detailed breakdown of backgrounds from diﬀerent sources. Events observed in the b jet pair and Z+jet mass plane, together with the predictions from background processes, are plotted in ﬁgure 6. The b jet pair and Z + jet mass distributions are shown separately in ﬁgure 7. Upper limits are calculated on the ratio of the measured Θ0 pair production cross sec- tion times branching fraction B(Θ0 →Zg) × B(Z →ℓℓ) × B(Θ0 →bb) × 2, to the theoreti- cally expected cross section times branching fraction, for each search region separately. We have used as a benchmark the case where B(Θ0 →bb) = B(Θ0 →Zg)+B(Θ0 →γg) = 0.5. The resulting limits placed on octo-triplet particle production cross section and mass can be scaled for diﬀerent choices of B(Θ0 →bb) and B(Θ0 →Zg). The results in the elec- tron and muon channels are consistent and therefore combined. A modiﬁed-frequentist approach, CLs, is used to calculate the limits [50, 51]. For each mass point, pseudo- – 11 – Figure 7. The b jet pair mass distributions for three diﬀerent ranges of Z+jet mass in the electron channel (top left) and the muon channel (top right); Z + jet mass distributions for three diﬀerent ranges of b jet pair mass in the electron channel (bottom left) and the muon channel (bottom right). The plotted regions correspond to three of the search regions. Predicted signal distributions are overlaid. The shaded band represents the statistical uncertainty combined with the systematic uncertainty in the simulated samples. JHEP09(2015)201 Figure 7. The b jet pair mass distributions for three diﬀerent ranges of Z+jet mass in the electron channel (top left) and the muon channel (top right); Z + jet mass distributions for three diﬀerent ranges of b jet pair mass in the electron channel (bottom left) and the muon channel (bottom right). The plotted regions correspond to three of the search regions. Predicted signal distributions are overlaid. The shaded band represents the statistical uncertainty combined with the systematic uncertainty in the simulated samples. 6 Results experiments are run for the signal plus background hypothesis, and for the background only hypothesis. The systematic uncertainties discussed in section 5, and their correla- tions, are included in the limit determinations through a set of nuisance parameters. The results are shown, in ﬁgure 8, as a 95% conﬁdence level (CL) observed and expected limit on σ ×B(Θ0 →Zg)×B(Z →ℓℓ)×B(Θ0 →bb)×2, as a function of Θ0 mass. For the case where mG′ = 2.3mΘ0, this result excludes Θ0 masses below 623 GeV at 95% CL, with an expected exclusion of 639 GeV. For the case where mG′ = 5mΘ0, Θ0 masses below 426 GeV are excluded at 95% CL, with an expected exclusion of 439 GeV. These plots also include the theoretical predictions, with the band indicating the uncertainty in the signal due to the PDF uncertainty. 7 Summary A search for pair production of neutral color-octet weak-triplet scalar particles (Θ0) has been performed based on processes where one Θ0 decays to a pair of b quark jets and the – 12 – mΘ0 [GeV] Signal Background Observed mG′ = 2.3mΘ0 mG′ = 5mΘ0 Electron channel 217 2110 ± 70 ± 290 1110 ± 40 ± 160 358 ± 15 ± 81 336 304 448 ± 12 ± 70 188 ± 6 ± 31 123 ± 6 ± 27 115 391 119 ± 3 ± 22 38.6 ± 1.3 ± 7.4 42.3 ± 3.0 ± 11.0 59 478 35.8 ± 1.0 ± 6.6 9.45 ± 0.32 ± 1.90 18.9 ± 2.7 ± 6.4 24 565 11.7 ± 0.3 ± 2.4 2.37 ± 0.09 ± 0.53 8.14 ± 1.94 ± 3.40 7 652 3.96 ± 0.13 ± 0.86 0.72 ± 0.03 ± 0.17 3.35 ± 0.82 ± 1.50 4 739 1.42 ± 0.06 ± 0.32 0.23 ± 0.01 ± 0.05 1.02 ± 0.31 ± 0.51 1 826 0.56 ± 0.02 ± 0.13 0.08 ± 0.02 0.66 ± 0.33 ± 0.36 0 913 0.24 ± 0.01 ± 0.06 0.03 ± 0.01 0.31 ± 0.14 ± 0.19 0 Muon channel 217 2360 ± 70 ± 310 1170 ± 40 ± 160 348 ± 11 ± 74 355 304 486 ± 12 ± 74 185 ± 6 ± 31 126 ± 7 ± 27 127 391 118 ± 3 ± 20 38.7 ± 1.2 ± 7.3 44.8 ± 3.1 ± 11.3 39 478 38.7 ± 1.0 ± 7.4 9.58 ± 0.32 ± 1.90 17.2 ± 1.9 ± 5.7 15 565 13.1 ± 0.4 ± 2.7 2.70 ± 0.09 ± 0.58 9.52 ± 1.40 ± 3.80 8 652 4.58 ± 0.14 ± 0.99 0.74 ± 0.03 ± 0.17 3.25 ± 0.84 ± 1.30 5 739 1.63 ± 0.07 ± 0.36 0.24 ± 0.01 ± 0.06 1.48 ± 0.49 ± 0.58 3 826 0.69 ± 0.03 ± 0.16 0.08 ± 0.02 0.32 ± 0.17 ± 0.14 2 913 0.26 ± 0.01 ± 0.06 0.03 ± 0.01 0.14 ± 0.14 ± 0.09 1 Table 2. The number of events after ﬁnal selection for the signal, total background, and ob- served data, together with the statistical and systematic uncertainty. For the entries where a single uncertainty is shown, the statistical uncertainty is negligible. JHEP09(2015)201 Table 2. 7 Summary The number of events after ﬁnal selection for the signal, total background, and ob- served data, together with the statistical and systematic uncertainty. For the entries where a single uncertainty is shown, the statistical uncertainty is negligible. other to a Z boson plus a jet, with the Z boson decaying to a pair of electrons or muons. This analysis is based on data collected with the CMS experiment in proton-proton collisions at √s = 8 TeV, corresponding to an integrated luminosity of 19.7 fb−1. The number of observed events is found to be in agreement with the standard model prediction. The CLs method is used to set a 95% conﬁdence level limit on the cross section of octo-triplet particles, assuming B(Θ0 →bb) = 0.5, with the remaining Θ0 branching fraction shared between Zg and γg. By comparing the theoretical predictions of the octo-triplet model and the observed limits, masses of Θ0 below 623 GeV for mG′ = 2.3mΘ0, and below 426 GeV for mG′ = 5mΘ0, are excluded at 95% conﬁdence level. These are the ﬁrst direct experimental bounds on the Θ0 production model. other to a Z boson plus a jet, with the Z boson decaying to a pair of electrons or muons. This analysis is based on data collected with the CMS experiment in proton-proton collisions at √s = 8 TeV, corresponding to an integrated luminosity of 19.7 fb−1. The number of observed events is found to be in agreement with the standard model prediction. The CLs method is used to set a 95% conﬁdence level limit on the cross section of octo-triplet particles, assuming B(Θ0 →bb) = 0.5, with the remaining Θ0 branching fraction shared between Zg and γg. By comparing the theoretical predictions of the octo-triplet model and the observed limits, masses of Θ0 below 623 GeV for mG′ = 2.3mΘ0, and below 426 GeV for mG′ = 5mΘ0, are excluded at 95% conﬁdence level. These are the ﬁrst direct experimental bounds on the Θ0 production model. 7 Summary – 13 – mΘ0 [GeV] Z + jets tt and tW Diboson Electron channel 217 256 ± 15 ± 79 91.3 ± 2.2 ± 18.0 11.1 ± 0.9 ± 2.2 304 90.5 ± 5.7 ± 26.0 27.5 ± 1.2 ± 5.3 4.55 ± 0.60 ± 0.90 391 33.6 ± 2.9 ± 11.0 6.55 ± 0.60 ± 1.30 2.10 ± 0.35 ± 0.42 478 16.9 ± 2.7 ± 6.4 1.24 ± 0.25 ± 0.24 0.78 ± 0.21 ± 0.16 565 7.36 ± 1.90 ± 3.30 0.36 ± 0.11 ± 0.07 0.42 ± 0.17 ± 0.08 652 3.08 ± 0.81 ± 1.50 0.04 ± 0.03 ± 0.01 0.23 ± 0.12 ± 0.05 739 1.02 ± 0.31 ± 0.51 < 0.04 < 0.01 826 0.65 ± 0.33 ± 0.36 < 0.04 0.01 ± 0.01 913 0.27 ± 0.13 ± 0.19 < 0.04 0.04 ± 0.04 ± 0.01 Muon channel 217 244 ± 10 ± 72 91.5 ± 2.2 ± 18.0 12.7 ± 1.0 ± 2.4 304 92.2 ± 7.2 ± 27.0 28.7 ± 1.2 ± 5.5 5.57 ± 0.59 ± 1.10 391 35.1 ± 3.0 ± 11.0 7.51 ± 0.62 ± 1.40 2.20 ± 0.36 ± 0.42 478 15.0 ± 1.8 ± 5.7 1.20 ± 0.26 ± 0.23 1.00 ± 0.24 ± 0.19 565 8.89 ± 1.40 ± 3.80 0.36 ± 0.13 ± 0.07 0.26 ± 0.11 ± 0.05 652 3.03 ± 0.84 ± 1.30 0.08 ± 0.07 ± 0.01 0.14 ± 0.05 ± 0.03 739 1.27 ± 0.48 ± 0.58 0.12 ± 0.07 ± 0.02 0.09 ± 0.04 ± 0.02 826 0.23 ± 0.16 ± 0.14 0.05 ± 0.05 ± 0.01 0.05 ± 0.03 ± 0.01 913 0.14 ± 0.14 ± 0.09 < 0.05 < 0.05 Table 3. The number of background events, from all sources, after ﬁnal selection, together wit the statistical and systematic uncertainty. For the entries where a single uncertainty is shown, th systematic uncertainty is negligible. JHEP09(2015)201 Table 3. The number of background events, from all sources, after ﬁnal selection, together with the statistical and systematic uncertainty. For the entries where a single uncertainty is shown, the systematic uncertainty is negligible. Acknowledgments We congratulate our colleagues in the CERN accelerator departments for the excellent performance of the LHC and thank the technical and administrative staﬀs at CERN and at other CMS institutes for their contributions to the success of the CMS eﬀort. In ad- dition, we gratefully acknowledge the computing centers and personnel of the Worldwide LHC Computing Grid for delivering so eﬀectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construction and operation of the LHC and the CMS detector provided by the following funding agencies: BMWFW and FWF (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MoST, and NSFC (China); COL- – 14 – [GeV] 0 Θ m 200 300 400 500 600 700 800 900 Br [pb] × σ 95% CL limit on -4 10 -3 10 -2 10 -1 10 1 10 0 Θ = 2.3 m G’ ), with m b (Zg)(b → 0 Θ 0 Θ → pp ) = 0.5 b b → 0 Θ B( g) = 0.5 γ → 0 Θ Zg) + B( → 0 Θ B( Expected limit σ 1 ± Expected σ 2 ± Expected Observed limit Color-Octet (with PDF uncertainty) (8 TeV) -1 19.7 fb CMS [GeV] 0 Θ m 200 300 400 500 600 700 800 900 Br [pb] × σ 95% CL limit on -4 10 -3 10 -2 10 -1 10 1 10 0 Θ = 5 m G’ ), with m b (Zg)(b → 0 Θ 0 Θ → pp ) = 0.5 b b → 0 Θ B( g) = 0.5 γ → 0 Θ Zg) + B( → 0 Θ B( Expected limit σ 1 ± Expected σ 2 ± Expected Observed limit Color-Octet (with PDF uncertainty) (8 TeV) -1 19.7 fb CMS Figure 8. The 95% CL expected and observed upper limits on the cross section times branching fraction, σ × B(Θ0 →Zg) × B(Z →ℓℓ) × B(Θ0 →bb) × 2, as a function of Θ0 mass, for the case where mG′ = 2.3mΘ0 (left) and mG′ = 5mΘ0 (right) with the band on the color octet theoretical prediction indicating uncertainty in the signal yield due to PDF. These results assume B(Θ0 →bb) = 0.5 and B(Θ0 →Zg) + B(Θ0 →γg) = 0.5. Acknowledgments CIENCIAS (Colombia); MSES and CSF (Croatia); RPF (Cyprus); MoER, ERC IUT and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NIH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Repub- lic of Korea); LAS (Lithuania); MOE and UM (Malaysia); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Dubna); MON, RosAtom, RAS and RFBR (Rus- sia); MESTD (Serbia); SEIDI and CPAN (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEPCenter, IPST, STAR and NSTDA (Thailand); TUBITAK and TAEK (Turkey); NASU and SFFR (Ukraine); STFC (United Kingdom); DOE and NSF (U.S.A.). CIENCIAS (Colombia); MSES and CSF (Croatia); RPF (Cyprus); MoER, ERC IUT and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); OTKA and NIH (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Repub- lic of Korea); LAS (Lithuania); MOE and UM (Malaysia); CINVESTAV, CONACYT, SEP, and UASLP-FAI (Mexico); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portugal); JINR (Dubna); MON, RosAtom, RAS and RFBR (Rus- sia); MESTD (Serbia); SEIDI and CPAN (Spain); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEPCenter, IPST, STAR and NSTDA (Thailand); TUBITAK and TAEK (Turkey); NASU and SFFR (Ukraine); STFC (United Kingdom); DOE and NSF (U.S.A.). Individuals have received support from the Marie-Curie program and the European Re- search Council and EPLANET (European Union); the Leventis Foundation; the A. P. Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Oﬃce; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT- Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOMING PLUS program of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund; the Compagnia di San Paolo (Torino); the Consorzio per la Fisica (Trieste); MIUR project 20108T4XTM (Italy); the Thalis and Aristeia programs coﬁnanced by EU-ESF and the Greek NSRF; and the National Priorities Research Program by Qatar National Research Fund. Individuals have received support from the Marie-Curie program and the European Re- search Council and EPLANET (European Union); the Leventis Foundation; the A. P. Acknowledgments [GeV] 0 Θ m 200 300 400 500 600 700 800 900 Br [pb] × σ 95% CL limit on -4 10 -3 10 -2 10 -1 10 1 10 0 Θ = 2.3 m G’ ), with m b (Zg)(b → 0 Θ 0 Θ → pp ) = 0.5 b b → 0 Θ B( g) = 0.5 γ → 0 Θ Zg) + B( → 0 Θ B( Expected limit σ 1 ± Expected σ 2 ± Expected Observed limit Color-Octet (with PDF uncertainty) (8 TeV) -1 19.7 fb CMS [GeV] 0 Θ m 200 300 400 500 600 700 800 900 Br [pb] × σ 95% CL limit on -4 10 -3 10 -2 10 -1 10 1 10 0 Θ = 5 m G’ ), with m b (Zg)(b → 0 Θ 0 Θ → pp ) = 0.5 b b → 0 Θ B( g) = 0.5 γ → 0 Θ Zg) + B( → 0 Θ B( Expected limit σ 1 ± Expected σ 2 ± Expected Observed limit Color-Octet (with PDF uncertainty) (8 TeV) -1 19.7 fb CMS JHEP09(2015)201 Figure 8. The 95% CL expected and observed upper limits on the cross section times branching fraction, σ × B(Θ0 →Zg) × B(Z →ℓℓ) × B(Θ0 →bb) × 2, as a function of Θ0 mass, for the case where mG′ = 2.3mΘ0 (left) and mG′ = 5mΘ0 (right) with the band on the color octet theoretical prediction indicating uncertainty in the signal yield due to PDF. These results assume B(Θ0 →bb) = 0.5 and B(Θ0 →Zg) + B(Θ0 →γg) = 0.5. 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Acknowledgments Sloan Foundation; the Alexander von Humboldt Foundation; the Belgian Federal Science Policy Oﬃce; the Fonds pour la Formation `a la Recherche dans l’Industrie et dans l’Agriculture (FRIA-Belgium); the Agentschap voor Innovatie door Wetenschap en Technologie (IWT- Belgium); the Ministry of Education, Youth and Sports (MEYS) of the Czech Republic; the Council of Science and Industrial Research, India; the HOMING PLUS program of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund; the Compagnia di San Paolo (Torino); the Consorzio per la Fisica (Trieste); MIUR project 20108T4XTM (Italy); the Thalis and Aristeia programs coﬁnanced by EU-ESF and the Greek NSRF; and the National Priorities Research Program by Qatar National Research Fund. Open Access. 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Beluﬃ4, O. Bondu, G. Bruno, R. Castello, A. Caudron, L. Ceard, G.G. Da Silveira, C. Delaere, D. Favart, L. Forthomme, A. Giammanco5, J. Hollar, A. Jafari, P. Jez, M. Komm, V. Lemaitre, A. Mertens, C. Nuttens, L. Perrini, A. Pin, K. Piotrzkowski, A. Popov6, L. Quertenmont, M. Selvaggi, M. Vidal Marono Universit´e de Mons, Mons, Belgium N. Beliy, T. Caebergs, G.H. Hammad Universit´e de Mons, Mons, Belgium N. Beliy, T. Caebergs, G.H. Hammad Centro Brasileiro de Pesquisas Fisicas, Rio de Janeiro, Brazil W.L. Ald´a J´unior, G.A. Alves, L. Brito, M. Correa Martins Junior, T. Dos Reis Martins, C. Hensel, C. Mora Herrera, A. Moraes, M.E. Pol, P. Rebello Teles – 19 – Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil E. Belchior Batista Das Chagas, W. Carvalho, J. Chinellato7, A. Cust´odio, E.M. Da Costa, D. De Jesus Damiao, C. De Oliveira Martins, S. Fonseca De Souza, L.M. Huertas Guativa, H. Malbouisson, D. Matos Figueiredo, L. Mundim, H. Nogima, W.L. Prado Da Silva, A. 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Finger Jr.10 – 20 – Academy of Scientiﬁc Research and Technology of the Arab Republic of Egypt, Egyptian Network of High Energy Physics, Cairo, Egypt A. Ali11,12, R. Aly13, S. Aly13, S. Elgammal12, A. Ellithi Kamel14, A. Lotfy15, M.A. Mahmoud15, R. Masod11, A. Radi12,11 National Institute of Chemical Physics and Biophysics, Tallinn, Estonia B. Calpas, M. Kadastik, M. Murumaa, M. Raidal, A. Tiko, C. Veelken Department of Physics, University of Helsinki, Helsinki, Finland P. Eerola, M. Voutilainen Helsinki Institute of Physics, Helsinki, Finland JHEP09(2015)201 J. H¨ark¨onen, V. Karim¨aki, R. Kinnunen, T. Lamp´en, K. Lassila-Perini, S. Lehti, T. Lind´en, P. Luukka, T. M¨aenp¨a¨a, J. Pekkanen, T. Peltola, E. Tuominen, J. Tuominiemi, E. Tuovi- nen, L. Wendland Lappeenranta University of Technology, Lappeenranta, Finland J. Talvitie, T. Tuuva DSM/IRFU, CEA/Saclay, Gif-sur-Yvette, France M. Besancon, F. Couderc, M. Dejardin, D. Denegri, B. Fabbro, J.L. Faure, C. Favaro, F. Ferri, S. 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Van Hove Centre de Calcul de l’Institut National de Physique Nucleaire et de Physique des Particules, CNRS/IN2P3, Villeurbanne, France S. Gadrat Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, N. Beaupere, C. Bernet9, G. Boudoul2, E. Bouvier, S. Brochet, C.A. Carrillo Montoya J Chasserat R Chierici D Contardo B Courbon P Depasse H El Mamouni Universit´e de Lyon, Universit´e Claude Bernard Lyon 1, CNRS-IN2P3, Institut de Physique Nucl´eaire de Lyon, Villeurbanne, France S. Beauceron, N. Beaupere, C. Bernet9, G. Boudoul2, E. Bouvier, S. Brochet, C.A. Carrillo Montoya, J. Chasserat, R. Chierici, D. Contardo, B. Courbon, P. Depasse, H. El Mamouni, J. Fan, J. Fay, S. Gascon, M. Gouzevitch, B. Ille, I.B. Laktineh, M. Lethuillier, L. Mirabito, A.L. Pequegnot, S. Perries, J.D. Ruiz Alvarez, D. Sabes, L. Sgandurra, V. Sordini, M. Vander Donckt, P. Verdier, S. Viret, H. Xiao – 21 – Institute of High Energy Physics and Informatization, Tbilisi State University, Tbilisi, Georgia D. Lomidze RWTH Aachen University, I. Physikalisches Institut, Aachen, Germany C. Autermann, S. Beranek, M. Edelhoﬀ, L. Feld, A. Heister, M.K. Kiesel, K. Klein, M. Lipinski, A. Ostapchuk, M. Preuten, F. Raupach, J. Sammet, S. Schael, J.F. Schulte, T. Verlage, H. Weber, B. Wittmer, V. Zhukov6 RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany M. Ata, M. Brodski, E. Dietz-Laursonn, D. Duchardt, M. Endres, M. Erdmann, S. Erdweg, T. Esch, R. Fischer, A. G¨uth, T. Hebbeker, C. Heidemann, K. Hoepfner, D. Klingebiel, S. Knutzen, P. Kreuzer, M. Merschmeyer, A. Meyer, P. Millet, M. Olschewski, K. Padeken, P. Papacz, T. Pook, M. Radziej, H. Reithler, M. Rieger, F. Scheuch, L. Sonnenschein, D. France Teyssier, S. Th¨uer JHEP09(2015)201 RWTH Aachen University, III. Physikalisches Institut B, Aachen, Germany V. Cherepanov, Y. Erdogan, G. Fl¨ugge, H. Geenen, M. Geisler, W. Haj Ahmad, F. Hoehle, B. Kargoll, T. Kress, Y. Kuessel, A. K¨unsken, J. Lingemann2, A. Nehrkorn, A. Nowack, I.M. Nugent, C. Pistone, O. Pooth, A. Stahl Deutsches Elektronen-Synchrotron, Hamburg, Germany Deutsches Elektronen-Synchrotron, Hamburg, Germany M. Aldaya Martin, I. Asin, N. Bartosik, O. Behnke, U. Behrens, A.J. Bell, K. Borras, A. Burgmeier, A. Cakir, L. Calligaris, A. Campbell, S. Choudhury, F. Costanza, C. Diez Pardos, G. Dolinska, S. Dooling, T. Dorland, G. Eckerlin, D. Eckstein, T. Eichhorn, G. Flucke, E. Gallo, J. Garay Garcia, A. Geiser, A. Gizhko, P. Gunnellini, J. Hauk, M. Hempel17, H. Jung, A. Kalogeropoulos, O. Karacheban17, M. Kasemann, P. Katsas, J. Kieseler, C. Kleinwort, I. Korol, W. Lange, J. Leonard, K. Lipka, A. Lobanov, W. Lohmann17, R. Mankel, I. Marﬁn17, I.-A. Melzer-Pellmann, A.B. Meyer, G. 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Hazi, P. Hidas, D. Horvath18, F. Sikler, V. Veszpremi, G. Vesztergombi19, A.J. Zsigmond Institute of Nuclear Research ATOMKI, Debrecen, Hungary N. Beni, S. Czellar, J. Karancsi20, J. Molnar, Z. Szillasi University of Debrecen, Debrecen, Hungary M. Bart´ok21, A. Makovec, P. Raics, Z.L. Trocsanyi, B. Ujvari National Institute of Science Education and Research, Bhubaneswar, India P. Mal, K. Mandal, N. Sahoo, S.K. Swain P. Mal, K. Mandal, N. Sahoo, S.K. Swain Panjab University, Chandigarh, India S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, N. Nishu, J.B. Singh, G. Walia University of Delhi, Delhi, India Ashok Kumar, Arun Kumar, A. Bhardwaj, B.C. Choudhary, R.B. Garg, A. Kumar, S Malhotra M Naimuddin K Ranjan R Sharma V Sharma S. Bansal, S.B. Beri, V. Bhatnagar, R. Chawla, R. Gupta, U.Bhawandeep, A.K. Kalsi, A. Kaur, M. Kaur, R. Kumar, A. Mehta, M. Mittal, N. Nishu, J.B. Singh, G. 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Sutar, N. Wickramage24 Majumder, K. Mazumdar, S. Mitra, G.B. Mohanty, B. Parida, T. Sarkar22, K. Sudhakar, Sur B Sutar N Wickramage24 G. Majumder, K. Mazumdar, S. Mitra, G.B. Mohanty, B. Parida, T. Sarkar22, K. Sudhakar, N. Sur, B. Sutar, N. Wickramage24 Indian Institute of Science Education and Research (IISER), Pune, India S. Sharma Institute for Research in Fundamental Sciences (IPM), Tehran, Iran Institute for Research in Fundamental Sciences (IPM), Tehran, Iran H. Bakhshiansohi, H. Behnamian, S.M. Etesami25, A. Fahim26, R. Goldouzian, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri, S. Paktinat Mehdiabadi, F. Rezaei Hosseinabadi, B. Safarzadeh27, M. Zeinali University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald JHEP09(2015)201 INFN Sezione di Bari a, Universit`a di Bari b, Politecnico di Bari c, Bari, Italy M. Abbresciaa,b, C. Calabriaa,b, C. Caputoa,b, S.S. Chhibraa,b, A. Colaleoa, D. Creanzaa,c, L. Cristellaa,b, N. De Filippisa,c, M. De Palmaa,b, L. Fiorea, G. Iasellia,c, G. Maggia,c, M. Maggia, G. Minielloa,b, S. Mya,c, S. Nuzzoa,b, A. Pompilia,b, G. Pugliesea,c, R. Radognaa,b, A. Ranieria, G. Selvaggia,b, A. Sharmaa, L. Silvestrisa,2, R. Vendittia,b, P. Verwilligena P. Verwilligena INFN Sezione di Bologna a, Universit`a di Bologna b, Bologna, Italy G. Abbiendia, C. Battilana2, A.C. Benvenutia, D. Bonacorsia,b, S. Braibant-Giacomellia,b, L. Brigliadoria,b, R. Campaninia,b, P. Capiluppia,b, A. Castroa,b, F.R. Cavalloa, G. Codispotia,b, M. Cuﬃania,b, G.M. Dallavallea, F. Fabbria, A. Fanfania,b, D. Fasanellaa,b, P. Giacomellia, C. Grandia, L. Guiduccia,b, S. Marcellinia, G. Masettia, A. Montanaria, F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b, R. Travaglinia,b F.L. Navarriaa,b, A. Perrottaa, A.M. Rossia,b, T. Rovellia,b, G.P. Sirolia,b, N. Tosia,b, R. Travaglinia,b INFN Sezione di Catania a, Universit`a di Catania b, CSFNSM c, Catania, Italy G. Cappelloa, M. Chiorbolia,b, S. Costaa,b, F. Giordanoa,c, R. Potenzaa,b, A. Tricomia,b, C. Tuvea,b INFN Sezione di Firenze a, Universit`a di Firenze b, Firenze, Italy G. Barbaglia, V. Ciullia,b, C. Civininia, R. D’Alessandroa,b, E. Focardia,b, S. Gonzia,b, V. Goria,b, P. Lenzia,b, M. Meschinia, S. Paolettia, G. Sguazzonia, A. Tropianoa,b, L. Viliania,b INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, F. Fabbri, D. Piccolo . Benussi, S. Bianco, F. Fabbri, D. Piccolo INFN Sezione di Genova a, Universit`a di Genova b, Genova, Italy V. Calvellia,b, F. Ferroa, M. Lo Veterea,b, E. Robuttia, S. Tosia,b INFN Sezione di Milano-Bicocca a, Universit`a di Milano-Bicocca b, Milano, Italy M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, R. Gerosaa,b, A. Ghezzia,b, P. Govonia,b, S M l ia R A M ia b B M hia b 2 D M a L M ia M.E. Dinardoa,b, S. Fiorendia,b, S. Gennaia, R. Gerosaa,b, A. Ghezzia,b, P. Govonia,b, S. Malvezzia, R.A. Manzonia,b, B. Marzocchia,b,2, D. Menascea, L. Moroni S. Malvezzia, R.A. Manzonia,b, B. Marzocchia,b,2, D. Menascea, L. Moronia, M. Paganonia,b, D. Pedrinia, S. Ragazzia,b, N. Redaellia, T. Tabarelli de Fatisa,b M. Paganonia,b, D. Pedrinia, S. Ragazzia,b, N. Redaellia, T. Tabarelli de Fatisa,b – 24 – INFN Sezione di Napoli a, Universit`a di Napoli ’Federico II’ b, Napoli, Italy, Universit`a della Basilicata c, Potenza, Italy, Universit`a G. Marconi d, Roma, Italy S. Buontempoa, N. Cavalloa,c, S. Di Guidaa,d,2, M. Espositoa,b, F. University College Dublin, Dublin, Ireland M. Felcini, M. Grunewald Fabozzia,c, A.O.M. Iorioa,b, G. Lanzaa, L. Listaa, S. Meolaa,d,2, M. Merolaa, P. Paoluccia,2, C. Sciaccaa,b, F. Thyssen INFN Sezione di Padova a, Universit`a di Padova b, Padova, Italy, Universit`a di Trento c, Trento, Italy P. Azzia,2, N. Bacchettaa, D. Biselloa,b, R. Carlina,b, A. Carvalho Antunes De Oliveiraa,b, P. Checchiaa, M. Dall’Ossoa,b,2, T. Dorigoa, F. Gasparinia,b, U. Gasparinia,b, A. Gozzelinoa, S. Lacapraraa, M. Margonia,b, A.T. Meneguzzoa,b, J. Pazzinia,b, N. Pozzobona,b, P. Ronchesea,b, M. Sgaravattoa, F. Simonettoa,b, E. Torassaa, M. Tosia,b, S. Vaninia,b, S. Venturaa, M. Zanetti, P. Zottoa,b, A. Zucchettaa,b,2, G. Zumerlea,b JHEP09(2015)201 Oliveira , , P. Checchia , M. Dall Osso , , , T. Dorigo , F. Gasparini , , U. Gasparini , , A. Gozzelinoa, S. Lacapraraa, M. Margonia,b, A.T. Meneguzzoa,b, J. Pazzinia,b, N. Pozzobona,b, P. Ronchesea,b, M. Sgaravattoa, F. Simonettoa,b, E. Torassaa, M. Tosia,b, S. Vaninia,b, S. Venturaa, M. Zanetti, P. Zottoa,b, A. Zucchettaa,b,2, G. Zumerlea,b INFN Sezione di Pavia a, Universit`a di Pavia b, Pavia, Italy A. Braghieria, M. Gabusia,b, A. Magnania, S.P. Rattia,b, V. Rea, C. Riccardia,b, P. Salvinia, I. Vaia, P. Vituloa,b INFN Sezione di Perugia a, Universit`a di Perugia b, Perugia, Italy L. Alunni Solestizia,b, M. Biasinia,b, G.M. Bileia, D. Ciangottinia,b,2, L. Fan`oa,b, P. Laricciaa,b, G. Mantovania,b, M. Menichellia, A. Sahaa, A. Santocchiaa,b, A. Spieziaa,b INFN Sezione di Pisa a, Universit`a di Pisa b, Scuola Normale Superiore di Pisa c, Pisa, Italy K. Androsova,28, P. Azzurria, G. Bagliesia, J. Bernardinia, T. Boccalia, G. Broccoloa K. Androsova,28, P. Azzurria, G. Bagliesia, J. Bernardinia, T. Boccalia, G. Broccoloa,c, R. Castaldia, M.A. Cioccia,28, R. Dell’Orsoa, S. Donatoa,c,2, G. Fedi, L. Fo`aa,c†, A. Giassia, M.T. Grippoa,28, F. Ligabuea,c, T. Lomtadzea, L. Martinia,b, A. Messineoa,b, F. Pallaa, A. Rizzia,b, A. Savoy-Navarroa,29, A.T. Serbana, P. Spagnoloa, P. Squillaciotia,28, R. Tenchinia, G. Tonellia,b, A. Venturia, P.G. Verdinia INFN Sezione di Roma a, Universit`a di Roma b, Roma, Italy L. Baronea,b, F. Cavallaria, G. D’imperioa,b,2, D. Del Rea,b, M. Diemoza, S. Gellia,b, C. Jordaa, E. Longoa,b, F. Margarolia,b, P. Meridiania, F. Michelia,b, G. Organtinia,b, R. Paramattia, F. Preiatoa,b, S. Rahatloua,b, C. Rovellia, F. Santanastasioa,b, L. Soﬃa,b, P. Traczyka,b,2 INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a d Piemonte Orientale c, Novara, Italy INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a del Piemonte Orientale c, Novara, Italy INFN Sezione di Torino a, Universit`a di Torino b, Torino, Italy, Universit`a del Piemonte Orientale c, Novara, Italy N. Amapanea,b, R. Arcidiaconoa,c, S. Argiroa,b, M. Arneodoa,c, R. Bellana,b, C. Biinoa, N. Cartigliaa, S. Casassoa,b, M. Costaa,b, R. Covarellia,b, A. Deganoa,b, N. Demariaa, L. Fincoa,b,2, C. Mariottia, S. Masellia, E. Migliorea,b, V. Monacoa,b, M. Musicha, M.M. Obertinoa,c, L. Pachera,b, N. Pastronea, M. Pelliccionia, G.L. Pinna Angionia,b, A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, A. Solanoa,b, A. Staianoa, U. Tamponia, P.P. Trapania,b A. Romeroa,b, M. Ruspaa,c, R. Sacchia,b, A. Solanoa,b, A. Staianoa, U. Tamponia, P.P. Trapania,b – 25 – INFN Sezione di Trieste a, Universit`a di Trieste b, Trieste, Italy S. Belfortea, V. Candelisea,b,2, M. Casarsaa, F. Cossuttia, G. Della Riccaa,b, B. Gobboa, C. La Licataa,b, M. Maronea,b, A. Schizzia,b, T. Umera,b, A. Zanettia Kangwon National University, Chunchon, Korea S. Chang, A. Kropivnitskaya, S.K. Nam Kyungpook National University, Daegu, Korea D.H. Kim, G.N. Kim, M.S. Kim, D.J. Kong, S. Lee, Y.D. Oh, A. Sakharov, D.C. Son Chonbuk National University, Jeonju, Korea H. Kim, T.J. Kim, M.S. Ryu JHEP09(2015)201 Chonnam National University, Institute for Universe and Elementary Particles, Kwangju, Korea Kwangju, Korea Kwangju, Korea S. Song S. Song Korea University, Seoul, Korea Warsaw, Poland G. Brona, K. Bunkowski, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Mis- iura, M. Olszewski, M. Walczak Laborat´orio de Instrumenta¸c˜ao e F´ısica Experimental de Part´ıculas, Lisboa, Portugal Korea University, Seoul, Korea S. Choi, Y. Go, D. Gyun, B. Hong, M. Jo, H. Kim, Y. Kim, B. Lee, K. Lee, K.S. Lee, S. Lee, S.K. Park, Y. Roh Seoul National University, Seoul, Korea H.D. Yoo University of Seoul, Seoul, Korea University of Seoul, Seoul, Korea M. Choi, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu University of Seoul, Seoul, Korea M. Choi, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu M. Choi, J.H. Kim, J.S.H. Lee, I.C. Park, G. Ryu Sungkyunkwan University, Suwon, Korea Y. Choi, Y.K. Choi, J. Goh, D. Kim, E. Kwon, J. Lee, I. Yu Sungkyunkwan University, Suwon, Korea Y. Choi, Y.K. Choi, J. Goh, D. Kim, E. Kwon, J. Lee, I. Yu Vilnius University, Vilnius, Lithuania A. Juodagalvis, J. Vaitkus Vilnius University, Vilnius, Lithuania A. Juodagalvis, J. Vaitkus National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia Z.A. Ibrahim, J.R. Komaragiri, M.A.B. Md Ali30, F. Mohamad Idris, W.A.T. Wan Abdullah Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico E. Casimiro Linares, H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-de La Cruz31, Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico E. Casimiro Linares, H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-de La Cruz31, A. Hernandez-Almada, R. Lopez-Fernandez, G. Ramirez Sanchez, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico S Carrillo Moreno F Vazquez Valencia Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico E. Casimiro Linares, H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-de La Cruz31, A. Hernandez-Almada, R. Lopez-Fernandez, G. Ramirez Sanchez, A. Sanchez-Hernandez Universidad Iberoamericana, Mexico City, Mexico S. Carrillo Moreno, F. Vazquez Valencia Benemerita Universidad Autonoma de Puebla, Puebla, Mexico S. Carpinteyro, I. Pedraza, H.A. Salazar Ibarguen Universidad Aut´onoma de San Luis Potos´ı, San Luis Potos´ı, Mexico A. Morelos Pineda – 26 – University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler, S. Reucroft National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, T. Khurshid, M. Shoaib National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski University of Auckland, Auckland, New Zealand D. Krofcheck University of Canterbury, Christchurch, New Zealand P.H. Butler, S. Reucroft University of Canterbury, Christchurch, New Zealand P.H. Butler, S. Reucroft National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, T. Khurshid, M. Shoaib National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan A. Ahmad, M. Ahmad, Q. Hassan, H.R. Hoorani, W.A. Khan, T. Khurshid, M. Shoaib National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski National Centre for Nuclear Research, Swierk, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. G´orski, M. Kazana, K. Nawrocki, K. Romanowska-Rybinska, M. Szleper, P. Zalewski JHEP09(2015)201 Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia A. Baskakov, A. Belyaev, E. Boos, M. Dubinin36, L. Dudko, A. Ershov, A. Gribushi A. Baskakov, A. Belyaev, E. Boos, M. Dubinin36, L. Dudko, A. Ershov, A. Gribushin, V. Klyukhin, O. Kodolova, I. Lokhtin, I. Myagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev , y , , , , , , V. Klyukhin, O. Kodolova, I. Lokhtin, I. Myagkov, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev Portugal P. Bargassa, C. Beir˜ao Da Cruz E Silva, A. Di Francesco, P. Faccioli, P.G. Ferreira Parracho, M. Gallinaro, L. Lloret Iglesias, F. Nguyen, J. Rodrigues Antunes, J. Seixas, O. Toldaiev, D. Vadruccio, J. Varela, P. Vischia Joint Institute for Nuclear Research, Dubna, Russia S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavi S. Afanasiev, P. Bunin, M. Gavrilenko, I. Golutvin, I. Gorbunov, A. Kamenev, V. Karjavin, V. Konoplyanikov, A. Lanev, A. Malakhov, V. Matveev32, P. Moisenz, V. Palichik, V. Perelygin, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, T. Toriashvili33, A. Zarubin V. Konoplyanikov, A. Lanev, A. Malakhov, V. Matveev32, P. Moisenz, V. Palichik, V. Perelygin, S. Shmatov, S. Shulha, N. Skatchkov, V. Smirnov, T. Toriashvili33, A. Zarubin Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia V. Golovtsov, Y. Ivanov, V. Kim34, E. Kuznetsova, P. Levchenko, V. Murzin, V. Oreshkin, I. Smirnov, V. Sulimov, L. Uvarov, S. Vavilov, A. Vorobyev V. Golovtsov, Y. Ivanov, V. Kim , E. Kuznetsova, P. Levchenko, V. Murzin, V. Oreshki I. Smirnov, V. Sulimov, L. Uvarov, S. Vavilov, A. Vorobyev Institute for Nuclear Research, Moscow, Russia Institute for Nuclear Research, Moscow, Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, D. Tlisov, A. Toropin Institute for Theoretical and Experimental Physics, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, E. Vlasov, A. Zhokin National Research Nuclear University ’Moscow Engineering Physics Insti- tute’ (MEPhI), Moscow, Russia A. Bylinkin P.N. Lebedev Physical Institute, Moscow, Russia P.N. Lebedev Physical Institute, Moscow, Russia V. Andreev, M. Azarkin35, I. Dremin35, M. Kirakosyan, A. Leonidov35, G. Mesyats, S.V. Rusakov, A. Vinogradov – 27 – Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, V. Kachanov, A. Kalinin, D. Konstantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, L. Tourtchanovitch, S. Troshin, N. Tyurin, State Research Center of Russian Federation, Institute for High Energy Physics, Protvino, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, V. Kachanov, A. Kalinin, D. Konstantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, L. Tourtchanovitch, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov I. Azhgirey, I. Bayshev, S. Bitioukov, V. Kachanov, A. Kalinin, D. Konstantinov, V. Krychkine, V. Petrov, R. Ryutin, A. Sobol, L. Tourtchanovitch, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov JHEP09(2015)201 University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia P. Adzic37, M. Ekmedzic, J. Milosevic, V. Rekovic Centro de Investigaciones Energ´eticas Medioambientales y Tec- nol´ogicas (CIEMAT), Madrid, Spain National Taiwan University (NTU), Taipei, Taiwan Chang, Y.H. Chang, Y.W. Chang, Y. Chao, K.F. Chen, P.H. Chen, C. Dietz, F. Fiori, P. Chang, Y.H. Chang, Y.W. Chang, Y. Chao, K.F. Chen, P.H. Chen, C. Dietz, F. Fiori, U. Grundler, W.-S. Hou, Y. Hsiung, Y.F. Liu, R.-S. Lu, M. Mi˜nano Moya, E. Petrakou, J.f. Tsai, Y.M. Tzeng, R. Wilken Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand Paul Scherrer Institut, Villigen, Switzerland Paul Scherrer Institut, Villigen, Switzerland Paul Scherrer Institut, Villigen, Switzerland W. Bertl, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, T. Rohe JHEP09(2015)201 W. Bertl, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, T. Rohe Institute for Particle Physics, ETH Zurich, Zurich, Switzerland F. Bachmair, L. B¨ani, L. Bianchini, M.A. Buchmann, B. Casal, G. Dissertori, M. Dittmar, M. Doneg`a, M. D¨unser, P. Eller, C. Grab, C. Heidegger, D. Hits, J. Hoss, G. Kasieczka, W. Lustermann, B. Mangano, A.C. Marini, M. Marionneau, P. Martinez Ruiz del Arbol, M. Masciovecchio, D. Meister, N. Mohr, P. Musella, F. Nessi-Tedaldi, F. Pandolﬁ, J. Pata, F. Pauss, L. Perrozzi, M. Peruzzi, M. Quittnat, M. Rossini, A. Starodumov42, M. Takahashi, V.R. Tavolaro, K. Theoﬁlatos, R. Wallny, H.A. Weber Universit¨at Z¨urich, Zurich, Switzerland T.K. Aarrestad, C. Amsler43, M.F. Canelli, V. Chiochia, A. De Cosa, C. Galloni, A. Hinz- mann, T. Hreus, B. Kilminster, C. Lange, J. Ngadiuba, D. Pinna, P. Robmann, F.J. Ronga, D. Salerno, S. Taroni, Y. Yang National Central University, Chung-Li, Taiwan National Central University, Chung-Li, Taiwan M. Cardaci, K.H. Chen, T.H. Doan, C. Ferro, M. Konyushikhin, C.M. Kuo, W. Lin, Y.J. Lu, R. Volpe, S.S. Yu M. Cardaci, K.H. Chen, T.H. Doan, C. Ferro, M. Konyushikhin, C.M. Kuo, W. Lin, Y.J. Lu, R. Volpe, S.S. Yu National Taiwan University (NTU), Taipei, Taiwan entro de Investigaciones Energ´eticas Medioambientales y Tec- ol´ogicas (CIEMAT), Madrid, Spain J. Alcaraz Maestre, E. Calvo, M. Cerrada, M. Chamizo Llatas, N. Colino, B. De La Cruz, A. Delgado Peris, D. Dom´ınguez V´azquez, A. Escalante Del Valle, C. Fernandez Bedoya, J.P. Fern´andez Ramos, J. Flix, M.C. Fouz, P. Garcia-Abia, O. Gonzalez Lopez, S. Goy Lopez, J.M. Hernandez, M.I. Josa, E. Navarro De Martino, A. P´erez-Calero Yzquierdo, J. Puerta Pelayo, A. Quintario Olmeda, I. Redondo, L. Romero, M.S. Soares Universidad Aut´onoma de Madrid, Madrid, Spain C. Albajar, J.F. de Troc´oniz, M. Missiroli, D. Moran , , p H. Brun, J. Cuevas, J. Fernandez Menendez, S. Folgueras, I. Gonzalez Caballero, E. Pa- lencia Cortezon, J.M. Vizan Garcia Instituto de F´ısica de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain J.A. Brochero Cifuentes, I.J. Cabrillo, A. Calderon, J.R. Casti˜neiras De Saa, J. Duarte Campderros, M. Fernandez, G. Gomez, A. Graziano, A. Lopez Virto, J. Marco, R. Marco, C. Martinez Rivero, F. Matorras, F.J. Munoz Sanchez, J. Piedra Gomez, T. Rodrigo, A.Y. Rodr´ıguez-Marrero, A. Ruiz-Jimeno, L. Scodellaro, I. Vila, R. Vilar Cortabitarte CERN, European Organization for Nuclear Research, Geneva, Switzerland D. Abbaneo, E. Auﬀray, G. Auzinger, M. Bachtis, P. Baillon, A.H. Ball, D. Bar- ney, A. Benaglia, J. Bendavid, L. Benhabib, J.F. Benitez, G.M. Berruti, G. Bianchi, P. Bloch, A. Bocci, A. Bonato, C. Botta, H. Breuker, T. Camporesi, G. Cerminara, S. Colafranceschi38, M. D’Alfonso, D. d’Enterria, A. Dabrowski, V. Daponte, A. David, M. De Gruttola, F. De Guio, A. De Roeck, S. De Visscher, E. Di Marco, M. Dobson, M. Dordevic, T. du Pree, N. Dupont-Sagorin, A. Elliott-Peisert, J. 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Grynyov National Scientiﬁc Center, Kharkov Institute of Physics and Technology, Kharkov, Ukraine JHEP09(2015)201 L. Levchuk, P. Sorokin L. Levchuk, P. Sorokin University of Bristol, Bristol, United Kingdom R. Aggleton, F. Ball, L. Beck, J.J. Brooke, E. Clement, D. Cussans, H. Flacher, J. Gold- stein, M. Grimes, G.P. Heath, H.F. Heath, J. Jacob, L. Kreczko, C. Lucas, Z. Meng, D.M. Newbold57, S. Paramesvaran, A. Poll, T. Sakuma, S. Seif El Nasr-storey, S. Senkin, D. Smith, V.J. Smith Rutherford Appleton Laboratory, Didcot, United Kingdom K.W. Bell, A. Belyaev58, C. Brew, R.M. Brown, D.J.A. Cockerill, J.A. Coughlan, K. Harder, S. Harper, E. Olaiya, D. Petyt, C.H. Shepherd-Themistocleous, A. Thea, I.R. Tomalin, T. Williams, W.J. Womersley, S.D. Worm Imperial College, London, United Kingdom M. Baber, R. Bainbridge, O. Buchmuller, A. Bundock, D. Burton, M. Citron, D. Colling, L. Corpe, N. Cripps, P. Dauncey, G. Davies, A. De Wit, M. Della Negra, P. Dunne, A. Elwood, W. Ferguson, J. 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S. Abdullin, M. Albrow, J. Anderson, G. Apollinari, L.A.T. Bauerdick, A. Beretvas, J. Berryhill, P.C. Bhat, G. Bolla, K. Burkett, J.N. Butler, H.W.K. Cheung, F. Chle- bana, S. Cihangir, V.D. Elvira, I. Fisk, J. Freeman, E. Gottschalk, L. Gray, D. Green, S. Gr¨unendahl, O. Gutsche, J. Hanlon, D. Hare, R.M. Harris, J. Hirschauer, B. Hoober- man, Z. Hu, S. Jindariani, M. Johnson, U. Joshi, A.W. Jung, B. Klima, B. Kreis, S. Kwan†, S. Lammel, J. Linacre, D. Lincoln, R. Lipton, T. Liu, R. Lopes De S´a, J. Lykken, K. Maeshima, J.M. Marraﬃno, V.I. Martinez Outschoorn, S. Maruyama, D. Mason, P. McBride, P. Merkel, K. Mishra, S. Mrenna, S. Nahn, C. Newman-Holmes, V. O’Dell, O. Prokofyev, E. Sexton-Kennedy, A. Soha, W.J. Spalding, L. Spiegel, L. Taylor, S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, C. Vernieri, M. Verzocchi, R. Vidal, A. Whitbeck, F. Yang, H. Yin JHEP09(2015)201 A. Whitbeck, F. Yang, H. Yin University of Florida, Gainesville, U.S.A. University of Florida, Gainesville, U.S.A. D. Acosta, P. Avery, P. Bortignon, D. Bourilkov, A. Carnes, M. Carver, D. Curry, S. Das, G.P. Di Giovanni, R.D. Field, M. Fisher, I.K. Furic, J. Hugon, J. Konigsberg, A. Korytov, T. Kypreos, J.F. Low, P. Ma, K. Matchev, H. Mei, P. Milenovic61, G. Mitselmakher, L. Muniz, D. Rank, L. Shchutska, M. Snowball, D. Sperka, S.J. Wang, J. Yelton Florida International University, Miami, U.S.A. S. Hewamanage, S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida International University, Miami, U.S.A. S. Hewamanage, S. Linn, P. Markowitz, G. Martinez, J.L. Rodriguez Florida State University, Tallahassee, U.S.A. A. Ackert, J.R. Adams, T. Adams, A. Askew, J. Bochenek, B. Diamond, J. Haas, S. Hagopian, V. Hagopian, K.F. Johnson, A. Khatiwada, H. Prosper, V. Veeraraghavan, M. Weinberg Florida Institute of Technology, Melbourne, U.S.A. V. Bhopatkar, M. Hohlmann, H. Kalakhety, D. Mareskas-palcek, T. Roy, F. Yumiceva University of Illinois at Chicago (UIC), Chicago, U.S.A. M.R. Adams, L. Apanasevich, D. Berry, R.R. Betts, I. Bucinskaite, R. Cavanaugh, O. Evdokimov, L. Gauthier, C.E. Gerber, D.J. Hofman, P. Kurt, C. O’Brien, I.D. Sandoval Gonzalez, C. Silkworth, P. Turner, N. Varelas, Z. Wu, M. Zakaria The University of Iowa, Iowa City, U.S.A. B. Bilki62, W. Clarida, K. Dilsiz, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khris- tenko, J.-P. Merlo, H. Mermerkaya63, A. Mestvirishvili, A. Moeller, J. Nachtman, H. Ogul, Y. Onel, F. Ozok52, A. Penzo, S. Sen64, C. Snyder, P. Tan, E. Tiras, J. Wetzel, K. Yi Johns Hopkins University, Baltimore, U.S.A. I. Anderson, B.A. Barnett, B. Blumenfeld, D. Fehling, L. Feng, A.V. Gritsan, P. Maksi- movic, C. Martin, K. Nash, M. Osherson, M. Swartz, M. Xiao, Y. Xin Massachusetts Institute of Technology, Cambridge, U.S.A. A. Apyan, R. Barbieri, A. Baty, K. Bierwagen, S. Brandt, W. Busza, I.A. Cali, L. Di Matteo, G. Gomez Ceballos, M. Goncharov, D. Gulhan, M. 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Kharchilava, A. Kumar, S. Rappoccio Northeastern University, Boston, U.S.A. y, , G. Alverson, E. Barberis, D. Baumgartel, M. Chasco, A. Hortiangtham, A. Massironi, D.M. Morse, D. Nash, T. Orimoto, R. Teixeira De Lima, D. Trocino, R.-J. Wang, D. Wood, J. Zhang G. Alverson, E. Barberis, D. Baumgartel, M. Chasco, A. Hortiangtham, A. Massiron D.M. Morse, D. Nash, T. Orimoto, R. Teixeira De Lima, D. Trocino, R.-J. Wang, D. Wood, J. Zhang Northwestern University, Evanston, U.S.A. K.A. Hahn, A. Kubik, N. Mucia, N. Odell, B. Pollack, A. Pozdnyakov, M. Schmitt, S. Stoynev, K. Sung, M. Trovato, M. Velasco, S. Won Northwestern University, Evanston, U.S.A. K.A. Hahn, A. Kubik, N. Mucia, N. Odell, B. Pollack, A. Pozdnyakov, M. Schmit S. Stoynev, K. Sung, M. Trovato, M. Velasco, S. Won University of Notre Dame, Notre Dame, U.S.A. A. Brinkerhoﬀ, N. Dev, M. Hildreth, C. Jessop, D.J. Karmgard, N. Kellams, K. Lannon, A. Brinkerhoﬀ, N. Dev, M. Hildreth, C. Jessop, D.J. Karmgard, N. Kellams, K. 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Woods †: Deceased 1: Also at Vienna University of Technology, Vienna, Austria 2: Also at CERN, European Organization for Nuclear Research, Geneva, Switzerland 3: Also at State Key Laboratory of Nuclear Physics and Technology, Peking University, Beijin China 4: Also at Institut Pluridisciplinaire Hubert Curien, Universit´e d Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 4: Also at Institut Pluridisciplinaire Hubert Curien, Universit´e de Strasbourg, Universit´e Haute Alsace Mulhouse, CNRS/IN2P3, Strasbourg, France 5: Also at National Institute of Chemical Physics and Biophysics, Tallinn, Estonia 5: Also at National Institute of Chemical Physics and Biophysics, Tallinn, Estonia 6: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 6: Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 7: Also at Universidade Estadual de Campinas, Campinas, Brazil 7: Also at Universidade Estadual de Campinas, Campinas, Brazil 8: Also at Centre National de la Recherche Scientiﬁque (CNRS) - IN2P3, Paris, France 8: Also at Centre National de la Recherche Scientiﬁque (CNRS) - IN2P3, Paris, France 9: Also at Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau, France 9: Also at Laboratoire Leprince-Ringuet, Ecole Polytechnique, IN2P3-CNRS, Palaiseau 10: Also at Joint Institute for Nuclear Research, Dubna, Russia 10: Also at Joint Institute for Nuclear Research, Dubna, Russia 1: Also at Ain Shams University, Cairo, Egypt 12: Now at British University in Egypt, Cairo, Eg 13: Now at Helwan University, Cairo, Egypt 14: Also at Cairo University, Cairo, Egypt 15: Now at Fayoum University, El-Fayoum, Egypt 16: Also at Universit´e de Haute Alsace, Mulhouse, France 16: Also at Universit´e de Haute Alsace, Mulhouse, France 17: Also at Brandenburg University of Technology, Cottbus, Germany 17: Also at Brandenburg University of Technology, Cottbus, Germany 18: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 18: Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungary 19: Also at E¨otv¨os Lor´and University, Budapest, Hungary 20: Also at University of Debrecen, Debrecen, Hungary 21: Also at Wigner Research Centre for Physics, Budapest, Hungary 21: Also at Wigner Research Centre for Physics, Budapest, Hungary 22: Also at University of Visva-Bharati, Santiniketan, India 22: Also at University of Visva-Bharati, Santiniketan, India 23: Now at King Abdulaziz University, Jeddah, Saudi Arabia 23: Now at King Abdulaziz University, Jeddah, Saudi Arabia 24: Also at University of Ruhuna, Matara, Sri Lanka 25: Also at Isfahan University of Technology, Isfahan, Iran – 35 – 26: Also at University of Tehran, Department of Engineering Science, Tehran, Iran 27: Also at Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran 28: Also at Universit`a degli Studi di Siena, Siena, Italy 29: Also at Purdue University, West Lafayette, U.S.A. University of Wisconsin, Madison, U.S.A. 30: Also at International Islamic University of Malaysia, Kuala Lumpur, Malaysia 31: Also at CONSEJO NATIONAL DE CIENCIA Y TECNOLOGIA, MEXICO, Mexico 32: Also at Institute for Nuclear Research, Moscow, Russia 32: Also at Institute for Nuclear Research, Moscow, Russia 33: Also at Institute of High Energy Physics and Informatization, Tbilisi State University, Tbilisi, Georgia 34: Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia JHEP09(2015)201 JHEP09(2015)201 35: Also at National Research Nuclear University ‘Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia 36: Also at California Institute of Technology, Pasadena, U.S.A. 37: Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia 38: Also at Facolt`a Ingegneria, Universit`a di Roma, Roma, Italy 39: Also at Scuola Normale e Sezione dell’INFN, Pisa, Italy 40: Also at University of Athens, Athens, Greece 41: Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Polan Also at Warsaw University of Technology, Insti 42: Also at Institute for Theoretical and Experimental Physics, Moscow, Russia 43: Also at Albert Einstein Center for Fundamental Physics, Bern, Switzerland 44: Also at Adiyaman University, Adiyaman, Turkey 45: Also at Mersin University, Mersin, Turkey 46: Also at Cag University, Mersin, Turkey 47: Also at Piri Reis University, Istanbul, Turkey Also at Gaziosmanpasa University, Tokat, Turk 49: Also at Anadolu University, Eskisehir, Turkey 50: Also at Ozyegin University, Istanbul, Turkey 51: Also at Izmir Institute of Technology, Izmir, Turkey 52: Also at Mimar Sinan University, Istanbul, Istanbul, Turkey 53: Also at Marmara University, Istanbul, Turkey 54: Also at Kafkas University, Kars, Turkey 55: Also at Yildiz Technical University, Istanbul, Turkey 56: Also at Kahramanmaras S¨utc¨u Imam University, Kahramanmaras, Turkey 56: Also at Kahramanmaras S¨utc¨u Imam University, Kahramanmaras, Turkey 57: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 57: Also at Rutherford Appleton Laboratory, Didcot, United Kingdom 58: Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom 58: Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom 59: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 59: Also at Instituto de Astrof´ısica de Canarias, La Laguna, Spain 60: Also at Utah Valley University, Orem, U.S.A. University of Wisconsin, Madison, U.S.A. 61: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 61: Also at University of Belgrade, Faculty of Physics and Vinca Institute of Nuclear Sciences, Belgrade, Serbia 62: Also at Argonne National Laboratory, Argonne, U.S 63: Also at Erzincan University, Erzincan, Turkey 64: Also at Hacettepe University, Ankara, Turkey 65: Also at Texas A&M University at Qatar, Doha, Qatar 66: Also at Kyungpook National University, Daegu, Korea – 36 –
https://openalex.org/W2064959694	http://www.scielo.br/pdf/codas/v26n2/2317-1782-codas-26-02-00138.pdf	English	null	Adaptation of the Child Development Clinical Risk Indicators instrument to retrospective parent report	CoDAS	2,014	cc-by	7,397	DOI: 10.1590/2317-1782/2014001IN DOI: 10.1590/2317-1782/2014001IN DOI: 10.1590/2317-1782/2014001IN Adaptation of the Child Development Clinical Risk Indicators instrument to retrospective parent report Original Article Artigo Original Study carried out at the Pontifícia Universidade Católica of São Paulo – PUC-SP – São Paulo (SP), Brazil. (1) Pontifícia Universidade Católica de São Paulo – PUC-SP – São Paulo (SP), Brazil. Financial support: Coordination for the Improvement of Higher Education Personnel (CAPES). Conflict of interests: nothing to declare. Adaptação do instrumento Indicadores Clínicos de Risco para o Desenvolvimento Infantil para questionário retrospectivo para pais Fernanda Prada Machado1 Ruth Ramalho Ruivo Palladino1 Maria Claudia Cunha1 Fernanda Prada Machado1 Ruth Ramalho Ruivo Palladino1 Maria Claudia Cunha1 ABSTRACT Purpose: To adapt the Child Development Clinical Risk Indicators (IRDI) instrument to retrospective report for parents of children from 3 to 7 years old and administer it in two groups. Methods: Participated on the study 72 subjects — parents of children aged from 2 years and 11 months to 7 years and 7 months — divided into two groups: Research Group and Control Group. The proposed modification to parent report transformed the 31 indicators (affirmations) into retrospective self-administered questions to parents, with responses in Likert scale. The IRDI-questionnaire was administered in the two groups. Results: The IRDI-questionnaire showed to be easy and quick to administer (average 15 minutes) and with low cost. The analysis of the questions obtained a good internal consistency value. The comparison between the groups by the parents’ answers to the 31 questions showed difference in 16 questions. Conclusion: The items of the IRDI-questionnaire concern the main Autism Spectrum Disorders risk signs pointed out in literature. The parents’ answers showed differences between the studied groups. Speech, Language and Hearing Sciences Autistic disorder Questionnaires Communication Language Fonoaudiologia Transtorno autístico Questionários Comunicação Linguagem RESUMO Objetivo: Adaptar o instrumento Indicadores Clínicos de Risco para o Desenvolvimento Infantil (IRDI) para questionário retrospectivo para pais de crianças de 3 a 7 anos e aplicá-lo em dois grupos de sujeitos. Métodos: Participaram do estudo 72 sujeitos — pais de crianças de 2 anos e 11 meses a 7 anos e 7 meses — divididos em dois grupos: Grupo Pesquisa e Grupo Controle. A proposta de modificação do instrumento para questionário transformou os 31 indicadores (afirmações) do primeiro em perguntas de caráter retrospectivo, dirigidas aos pais em formato autoaplicável, com respostas em escala Likert. O IRDI-questionário foi aplicado nos dois grupos estudados. Resultados: O IRDI-questionário mostrou-se de fácil aplicação, com rapidez no preenchimento (tempo médio de 15 minutos) e baixo custo. A análise das questões que compõem o instrumento revelou boa consistência interna. A comparação entre os grupos, segundo as respostas dos pais às 31 questões do IRDI-questionário, mostrou diferença em 16 delas. Conclusão: Foi possível verificar que os itens do questionário recobrem os principais sinais de risco para Transtorno do Espectro do Autismo apontados na literatura. Além disso, as respostas dos pais assinalaram que houve diferença entre os grupos estudados. Correspondence address: Fernanda Prada Machado Graduate Program in Speech Language Pathology and Audiology, Rua Monte Alegre, 499, São Paulo (SP), Brazil, CEP: 05014-000. E-mail: fernandapradamachado@gmail.com Correspondence address: Fernanda Prada Machado Graduate Program in Speech Language Pathology and Audiology, Rua Monte Alegre, 499, São Paulo (SP), Brazil, CEP: 05014-000. E-mail: fernandapradamachado@gmail.com Received: 07/11/2013 Casuistry The sample was obtained by convenience, with consecutive selection. A total of 72 individuals took part in the research: 65 mothers and 7 parents of children aged from 2 years and 11 months to 7 years and 7 months. The significant results of intensive therapeutic interven- tions with very young children have encouraged this move- ment, which led to the need for use of diagnostic, assessment, and identification tools as a starting point for a differentiated prognosis(5,11-13). The subjects were divided into Study Group (SG) and Control Group (CG), to preserve the validity analysis and the reliability of the instrument. Articulating the academic perspective of research and pro- cedures in Public Health, it is recommended to identify chil- dren who are at risk for an eventual diagnosis of ASD using effective and low-cost methods. The main strategy toward this purpose, according to several authors, would be the use of an instrument in the format of a retrospective question- naire for parents(14-16). The SG consisted of 34 mothers (94.4%) and 2 fathers (5.6%) of children who were selected according to the inclu- sion criteria described next, being 29 of them males (80.6%) and 7 of them females (19.4%), who received autism ambu- latory care from the Institute of Psychiatry of the Hospital das Clínicas of the School of Medicine, Universidade de São Paulo (HC-FMUSP), and in private practices, aged between 35 and 91 months (2 years and 11 months and 7 years and 7 months). In Brazil, it is still very common for children to get to speech language pathology and audiology therapy at around 3–4 years of age, with recurrent complaints of delay in lan- guage development, often with suspected hearing loss, with- out any prior research or diagnosis for ASD having been performed(17). Inclusion criteria were children with diagnosed ASD, according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria(19) (American Psychiatric Association, 1995), and the absence of genetic, metabolic, and/or neurological disorders diagnosis, with the “IRDI ques- tionnaire” answered by the parents, and the informed consent signed by them. In Brazil, the aim at the development of instruments for early detection of problems in child development, a multi- centered study (in the period of 2000–2008), with the support of the Ministry of Health, the São Paulo Research Foundation and the National Council for Scientific and Technological Development, was carried out. INTRODUCTION This study aimed at articulating the important results of the research developed with IRDI in health to the interven- tions made by Brazilian speech-language pathologists, who often meet — as evidenced by the clinical experience — with children showing signs of risk for ASD and having difficulty in accessing free-of-charge clinical tools in Brazil. The infantile autism has been studied by various areas of knowledge since its first description in 1943 by Léo Kanner. Since then, its definition has undergone several modifications and, currently, the great variability in clinical signs shown by this group of individuals made the use of the term “autism spec- trum disorders” (ASD) more appropriate(1). In this sense, it seems promising to investigate the appli- cability of the IRDI instrument in the form of a questionnaire for parents, which is the theme of this research. It is known that language difficulties are an important dimen- sion of this clinical condition, next to impairments in social interaction and behavior(1-4). Such considerations support what is proposed by this study, namely to adapt the IRDI instrument into a retrospective ques- tionnaire for parents of children from 3 to 7 years of age and apply it, for the sake of comparison, in two groups of partici- pants (affected and not-affected by ASD). In general, the clinical signs of ASD can be observed in very young children, since the signs involve changes in skills that are typically developed in the first 2 years of life(5-8). In this context, some variability in the concerns expressed by parents occurs; however, complaints about the delay in the development of oral language are among the most common and consensual ones(4,9,10). METHODS This study was approved by the ethics committee in research of the university in which it was developed (protocol no. 117/2011). Family concerns in relation to the language development of these children is one of the reasons why the speech language pathologist is, in many cases, the first professional sought by the family, even before the diagnosis of the ASD has been advised or investigated. Thus, the relevance of speech language pathology and audiology in identifying possible risk for ASD is evident.i Accepted: 03/13/2014 CoDAS 2014;26(2):138-47 139 Adaptation of the IRDI for parents’ questionnaire Statistical analysis After the completion of this adaptation, it was given to two professionals, with extensive experience in administering the IRDI in its original format, to assess the IRDI questionnaire and suggest the necessary adjustments (which were not sig- nificant). Afterwards, the IRDI questionnaire was applied in both studied groups. A descriptive analysis of the data using absolute and rela- tive frequencies, measures of central tendency (average and median), and dispersion (standard deviation (SD), minimum and maximum) was conducted. To assess the homogeneity between the groups (CG and SG), we used the association of the χ2 test and the comparison of the Student’s t-test averages. In the internal consistency analysis of the IRDIquestionnaire, the Cronbach’s a was used. Statistical significance was taken on a significance level of 5% (p≤0.05). Adaptation of the instrument After the selection of the classrooms where the question- naires would be handed in (according to the age groups pre- viously established as inclusion criteria for the study), it was verified with the direction of the EMEI if there were com- plaints regarding any students, as to their development both in general terms and specifically about language and/or behavior. Students with complaints of this nature from the school were excluded from the study. The IRDI questionnaire is introduced by considerations about the nature of the questions and instructions regarding its filling out. Then, information regarding both the informant and the child is requested. The 31 statements of the original instrument (IRDI) were transformed into retrospective questions, directed toward par- ents. For this, only the verbal tense (from present to simple past) was modified, in order to preserve their original contents. On the date of the meeting with the parents, the researcher provided a brief explanation about the study and investi- gated, at the time the questionnaires were handed in, if the parents had any complaints as to the development of their children regarding their development in general terms, and in language and/or behavior, so as to exclude those with complaints. The answers, which in the original instrument were “absent,” “present,” and “unverified,” in the new format (questionnaire) were placed on a Likert scale that quantified the behaviors into five categories: “never,” “rarely,” “sometimes,” “often,” and “always.” In addition to these categories, the option “do not remember” was also included, because it was a retrospec- tive questionnaire, and therefore, based solely on the memory of parents about the early development of their children. The Likert scale was the chosen one, consisting of categories that are linked in a continuum, so as not to unduly constrain the answers, in favor of the reliability of the instrument. An explanation about the contents of the IRDIquestionnaire, which was handed in to one of the parents (the one who would volunteer so) to be filled out by, was also performed. The aver- age filling out time was 15 minutes. Casuistry From this study an instrument has emerged, which comprises the Clinical Risk Indicators for Child Development (Indicadores Clínicos de Risco para o Desenvolvimento Infantil, IRDI), consisting of 31 indicators observable in the first 18 months of the child’s life, whose use is indicated for pediatricians and other health profes- sionals(18). It is noteworthy that the IRDI is not a specific instrument for ASD, but for the problems in child develop- ment broadly. The CG consisted of 31 mothers (86.1%) and 5 fathers (13.9%) of children selected following the criteria described as follows: 23 male children (63.9%) and 13 female ones (36.1%), aged between 39 and 82 months (3 years and 3 months and 6 years and 10 months), with typical development and who attended the Public School of Early Childhood Education (EMEI) Curumim I, in Santana de Parnaíba. Inclusion criteria were children without complaints, by the parents and by the school, regarding their development and the absence of genetic, neurologic, and/or metabolic disorders CoDAS 2014;26(2):138-47 Machado FP, Palladino RRR, Cunha MC 140 diagnosis, with the “IRDI questionnaire” answered by the par- ents and the informed consent signed by them. in the multidisciplinary assessments. Once confirmed about the interest of the parents, a return would be scheduled for the evaluations, including those relating to this research. Administering the Clinical Risk Indicators for Child Development questionnaire in the Control Group Administering the Clinical Risk Indicators for Child Development questionnaire in the Control Group The adaptation of the IRDI instrument into the IRDI ques- tionnaire turned its 31 indicators (statements) into questions (ret- rospective ones), directed toward parents, in a self-report format. The adaptation procedure is described in the next section. After the paperwork needed for the authorization of the Education Department of the city of Santana de Parnaíba for carrying out of this research, the researcher attended the school, designated by the City Hall, in order to get in touch with the direction. It was agreed that the “IRDIquestionnaires” would be distributed by the researcher in PTA meetings, scheduled according to the school year’s calendar. Administering the Clinical Risk Indicators for Child Development questionnaire in the Study Group As part of the routine service of the Autism Clinic of the Institute of Psychiatry of the HC-USP, a team of researchers developed a protocol for multidisciplinary evaluations. Material On the scheduled date, the researcher would provide a brief explanation about the ethical issues contained in the informed consent and about the contents of IRDIquestionnaire, which was delivered to one of the parents (the one who vol- unteered so) so as to answer the questions. It is observed that the average time taken to fill out the questionnaire was 15 minutes. IRDI questionnaire: adapted IRDI into a retrospective ques- tionnaire for parents with scaled answers. Note: the IRDI instrument was validated in Brazil, consist- ing of 31 observable indicators in the first 18 months of the child’s life. Its application is given by observation, followed by the recording of the results into three categories: present, absent, or not verified indicators. The absence of at least two indicators indicates risk of development(14). Administering the Clinical Risk Indicators for Child Development questionnaire in the Control Group RESULTS For the purposes of this study, patients who attended the service for routine psychiatric care were approached by the research team to check if they had interest in participating The IRDIquestionnaire proved itself to be easily applied, quick to be filled out (15 minutes on average), and to have low cost. CoDAS 2014;26(2):138-47 Adaptation of the IRDI for parents’ questionnaire 141 Adaptation of the IRDI for parents’ questionnaire Up next, the results of its application in the two studied groups (SG and CG) are presented. After checking the homogeneity between the groups, the analysis of the internal consistency of the 31 questions of the instrument by Cronbach’s α, which had a value of 0.89, was performed; α value ≥0.6 was considered as satisfac- tory for internal consistency. Thus, it can be stated that the IRDIquestionnaire presented good internal consistency(20). The studied population comprised 72 children; 36 in each group. It is observed that there was a higher percentage of boys in both groups and that this distribution was homogeneous (p=0.114; Table 1). The average age of the children in the CG was 58.8 months (SD=9.2) and, for the children of the SG, it was 62.6 months (SD=15.0), presenting no difference between the groups (p=0.199). As to the parental educa- tional level, there was also no difference between the groups (p=0.690; Table 2). The comparison between the groups has shown differ- ences in 16 questions, which can be seen in Table 3. A higher proportion of children in the CG whose parents answered “always” to the question of whether there was an exchange of glances between the child and the mother, when compared to the answers of parents of children with ASD (p=0.006). According to parents, the likelihood of the child “always,” when requesting their mother, wait for their mother’s response was higher in the CG in relation to the SG (p=0.002). Regarding the age at the time of the diagnosis and the time elapsed between diagnosis and the questionnaire’s application in the SG, it appears that the average age of diagnosis is approximately 3 years and 6 months, ranging between 2 years and 5 months and 4 years and 9 months. In relation to the time elapsed, the average was 20 months (1 year and 8 months), with a minimum time less than 1 month and a maximum of 3 years and 10 months. RESULTS In the CG, there is a higher proportion of children whose mothers “always” perceived that some requests from the child could be a way to draw their attention, when compared to the answers of parents of children with ASD (p<0.001). The answers of the parents indicate that the probability of the child “always” actively seeking games and loving playfulness with their mother, during physical care pro- cedures, was higher in the CG when compared to that in the SG (p=0.005). The answers of the parents indicate that the probability of the child “always” actively seeking games and loving playfulness with their mother, during physical care pro- cedures, was higher in the CG when compared to that in the SG (p=0.005). According to parents, children in the CG are more likely to “always” show like or dislike toward something when compared to those in the SG (p=0.030). It is observed that in the CG the probability of mothers and children sharing a particular and private language between themselves was higher than that in the SG (p=0.023). It is also observed that the CG children are more likely to “make cute stunts” when compared to those of the SG (p<0.001). Likewise, from the parents’ answers, it is noticeable that the CG children are more likely to get the look of approval from the adult when compare to those with ASD (p<0.001). The parent responses indicate that 27.8% of the moth- ers of children with ASD “never” felt not obliged to ful- fill everything the child would ask for, whereas 8.3% of the mothers of children in the CG felt the same (p=0.037). The parent responses indicate that 27.8% of the moth- ers of children with ASD “never” felt not obliged to ful- fill everything the child would ask for, whereas 8.3% of the mothers of children in the CG felt the same (p=0.037). In the answers of the parents, it is still possible to see that the probability of the child looking with curiosity toward what interests the mother was higher in the CG (p<0.001). Therefore, in the comparison between the studied groups, through the IRDIquestionnaire, it was possible to distin- guish children with ASD from the ones presenting typical development, considering the 16 (of the 31) questions that were statistically significant. Table 2. RESULTS It is noteworthy that the IRDIquestionnaire was administered to some chil- dren concurrently with the diagnostic process; therefore, the minimum time spent between diagnosis and completion of the questionnaire was less than a month in these cases. It was also found that the CG children were more likely to react (smile, vocalize) when the mother or other per- son addressed them when compared to children with ASD (p<0.001). Children in the CG had a higher proportion in the reaction of “actively search for their mother’s look” in relation to those with ASD (p<0.001). In the same way, the proportion of children who would “always” ask for help from someone else instead of remaining passive was higher in the CG (p=0.031). Table 1. Number and percentual of children according to the study group, gender, and school education Variable Group p-value* Control Study n (%) n (%) Gender Male 23 (63.9) 29 (80.6) 0.114 Female 13 (36.1) 7 (19.4) Parents’ school education Incomplete grade school 3 (8.3) 6 (16.7) 0.690 Complete grade school 2 (5.6) 3 (8.3) Incomplete high school 3 (8.3) 2 (5.6) Complete high school 11 (30.6) 13 (36.1) Incomplete higher education 5 (13.9) 4 (11.1) Complete higher education/ college degree 7 (19.4) 6 (16.7) Incomplete graduate school 3 (8.3) 0 (0) Complete graduate school 2 (5.6) 2 (5.6) Informant Mother 31 (86.1) 34 (94.4) 0.429 Father 5 (13.9) 2 (5.6) Total 36 (100) 36 (100) χ2 test Table 2. Description of age at the time of the diagnosis and time elapsed between the diagnosis and the administration of the questionnaire, only for the Study Group Variable n Mean (SD) Median (Min–Max) Age at the time of the diagnosis 36 42.5 (7.5) 43.5 (29.0–57.0) Time elapsed between the diagnosis and the administration of the questionnaire 36 20.3 (15.0) 20.0 (0–46.0) Caption: SD = standard deviation; Min = minimum; Max = maximum Table 1. Number and percentual of children according to the study group, gender, and school education In the CG, there is a higher proportion of children whose mothers “always” perceived that some requests from the child could be a way to draw their attention, when compared to the answers of parents of children with ASD (p<0.001). RESULTS Description of age at the time of the diagnosis and time elapsed between the diagnosis and the administration of the questionnaire, only for the Study Group Variable n Mean (SD) Median (Min–Max) Age at the time of the diagnosis 36 42.5 (7.5) 43.5 (29.0–57.0) Time elapsed between the diagnosis and the administration of the questionnaire 36 20.3 (15.0) 20.0 (0–46.0) Caption: SD = standard deviation; Min = minimum; Max = maximum Table 2. Description of age at the time of the diagnosis and time elapsed between the diagnosis and the administration of the questionnaire, only for the Study Group In the answers of the parents, it is still possible to see that the probability of the child looking with curiosity toward what interests the mother was higher in the CG (p<0.001). Therefore, in the comparison between the studied groups, through the IRDIquestionnaire, it was possible to distin- guish children with ASD from the ones presenting typical development, considering the 16 (of the 31) questions that were statistically significant. CoDAS 2014;26(2):138-47 Machado FP, Palladino RRR, Cunha MC 142 Continues... Table 3. Number and percentage of children according to group and questions of the Clinical Risk Indicators for Child Development questionnaire (1–31) Variable Category Group p-value Control Study n % n % 1. When the child would cry or scream, did the mother know what they wanted? Always 9 (25.0) 13 (36.1) 0.072 Often 18 (50.0) 8 (22.2) Sometimes 7 (19.4) 10 (27.8) Rarely 0 (0.0) 4 (11.1) Never 1 (2.8) 1 (2.8) Do not remember 1 (2.8) 0 (0.0) 2. Would the mother speak to the child in a style directed particularly to them (“motherese”)? Always 15 (41.7) 10 (27.8) 0.737 Often 9 (25.0) 12 (33.3) Sometimes 8 (22.2) 8 (22.2) Rarely 3 (8.3) 3 (8.3) Never 1 (2.8) 2 (5.6) Do not remember 0 (0.0) 1 (2.8) 3. Would the child react to the motherese? Always 14 (38.9) 9 (25.0) 0.278 Often 9 (25.0) 6 (16.7) Sometimes 8 (22.2) 8 (22.2) Rarely 4 (11.1) 7 (19.4) Never 1 (2.8) 4 (11.1) Do not remember 0 (0.0) 2 (5.6) 4. Would the mother propose anything to the child and wait for their reaction? Always 13 (36.1) 12 (33.3) 0.779 Often 14 (38.9) 15 (41.7) Sometimes 7 (19.4) 6 (16.7) Rarely 1 (2.8) 2 (5.6) Never 0 (0.0) 1 (2.8) Do not remember 1 (2.8) 0 (0.0) 5. Continues... RESULTS Was there any exchange of looks (eye contact) between the child and the mother? Always 25 (69.4) 16 (44.4) 0.006 Often 10 (27.8) 5 (13.9) Sometimes 1 (2.8) 5 (13.9) Rarely 0 (0.0) 8 (22.2) Never 0 (0.0) 1 (2.8) Do not remember 0 (0.0) 1 (2.8) 6. Would the child start to differ day from night? Always 11 (30.6) 12 (33.3) 0.005 Often 12 (33.3) 4 (11.1) Sometimes 9 (25.0) 4 (11.1) Rarely 1 (2.8) 5 (13.9) Never 0 (0.0) 8 (22.2) Do not remember 3 (8.3) 3 (8.3) 7. Would the child use different signals in order to express their different needs? Always 9 (25.0) 6 (16.7) 0.100 Often 14 (38.9) 8 (22.2) Sometimes 10 (27.8) 10 (27.8) Rarely 2 (5.6) 4 (11.1) Never 0 (0.0) 6 (16.7) Do not remember 1 (2.8) 2 (5.6) 8. Would the child request their mother and make a pause in order to wait for the mother’s response? Always 12 (33.3) 5 (13.9) 0.002 Often 9 (25.0) 3 (8.3) Sometimes 10 (27.8) 6 (16.7) Rarely 2 (5.6) 12 (33.3) Never 1 (2.8) 8 (22.2) Do not remember 2 (5.6) 2 (5.6) 9. Would the mother address to the child using short sentences? Always 17 (47.2) 15 (41.7) 0.368 Often 11 (30.6) 17 (47.2) Sometimes 6 (16.7) 3 (8.3) Rarely 0 (0.0) 1 (2.8) Never 1 (2.8) 0 (0.0) Do not remember 1 (2.8) 0 (0.0) Table 3. Number and percentage of children according to group and questions of the Clinical Risk Indicators for Child Development questionnaire (1–31) of children according to group and questions of the Clinical Risk Indicators for Child Development questionnaire (1–31) ble 3. Number and percentage of children according to group and questions of the Clinical Risk Indicators for Child Deve CoDAS 2014;26(2):138-47 143 Adaptation of the IRDI for parents’ questionnaire Table 3. Continuation Continues... Variable Category Group p-value* Control Study n % n % 10. Would the child react (smile, vocalize) when addressed by the mother or by anybody else? Always 24 (66.7) 8 (22.2) <0.001 Often 12 (33.3) 10 (27.8) Sometimes 0 (0.0) 10 (27.8) Rarely 0 (0.0) 4 (11.1) Never 0 (0.0) 4 (11.1) Do not remember 0 (0.0) 0 (0.0) 11. Would the child actively search for the mother’s look? Continues... RESULTS Always 19 (52.8) 4 (11.1) <0.001 Often 14 (38.9) 12 (33.3) Sometimes 2 (5.6) 8 (22.2) Rarely 1 (2.8) 8 (22.2) Never 0 (0.0) 3 (8.3) Do not remember 0 (0.0) 1 (2.8) 12. Would the mother provide support to the initiatives of the child without sparing them of the effort? Always 17 (47.2) 11 (30.6) 0.080 Often 14 (38.9) 11 (30.6) Sometimes 5 (13.9) 8 (22.2) Rarely 0 (0.0) 4 (11.1) Never 0 (0.0) 0 (0.0) Do not remember 0 (0.0) 2 (5.6) 13. Would the child ask for the help of other people without falling into passivity? Always 8 (22.2) 2 (5.6) 0.031 Often 10 (27.8) 10 (27.8) Sometimes 11 (30.6) 6 (16.7) Rarely 2 (5.6) 12 (33.3) Never 4 (11.1) 5 (13.9) Do not remember 1 (2.8) 1 (2.8) 14. Would the mother notice that some requests made by the child could be a way of drawing her attention? Always 12 (33.3) 3 (8.3) <0.001 Often 18 (50.0) 4 (11.1) Sometimes 4 (11.1) 11 (30.6) Rarely 1 (2.8) 9 (25.0) Never 0 (0.0) 6 (16.7) Do not remember 1 (2.8) 3 (8.3) 15. During bodily care, would the child actively search for games and loving playfulness with the mother? Always 22 (61.1) 7 (19.4) 0.005 Often 7 (19.4) 7 (19.4) Sometimes 4 (11.1) 11 (30.6) Rarely 2 (5.6) 3 (8.3) Never 1 (2.8) 7 (19.4) Do not remember 0 (0.0) 1 (2.8) 16. Would the child manifest like or dislike toward anything? Always 12 (33.3) 10 (27.8) 0.030 Often 18 (50.0) 8 (22.2) Sometimes 5 (13.9) 12 (33.3) Rarely 1 (2.8) 4 (11.1) Never 0 (0.0) 2 (5.6) Do not remember 0 (0.0) 0 (0.0) 17. Do mother and child share a particular private language between themselves? Always 14 (38.9) 9 (25.0) 0.023 Often 12 (33.3) 6 (16.7) Sometimes 8 (22.2) 8 (22.2) Rarely 1 (2.8) 3 (8.3) Never 0 (0.0) 9 (25.0) Do not remember 1 (2.8) 1 (2.8) 18. Would the child feel uncomfortable with people to whom they were not familiar with? Always 9 (25.0) 5 (13.9) 0.119 Often 6 (16.7) 6 (16.7) Sometimes 15 (41.7) 9 (25.0) Rarely 4 (11.1) 9 (25.0) Never 2 (5.6) 7 (19.4) Do not remember 0 (0.0) 0 (0.0) CoDAS 2014;26(2):138-47 Machado FP, Palladino RRR, Cunha MC 144 Table 3. Continuation Table 3. Continuation Continues... Variable Category Group p-value* Control Study n % n % 19. RESULTS Would the child possess favorite objects? Always 13 (36.1) 11 (30.6) 0.404 Often 9 (25.0) 6 (16.7) Sometimes 9 (25.0) 6 (16.7) Rarely 1 (2.8) 4 (11.1) Never 3 (8.3) 7 (19.4) Do not remember 1 (2.8) 2 (5.6) 20. Would the child “make cute stunts”? Always 14 (38.9) 4 (11.1) <0.001 Often 18 (50.0) 9 (25.0) Sometimes 4 (11.1) 14 (38.9) Rarely 0 (0.0) 4 (11.1) Never 0 (0.0) 4 (11.1) Do not remember 0 (0.0) 1 (2.8) 21. Would the child search for the adult’s look of approval? Always 9 (25.0) 3 (8.3) <0.001 Often 18 (50.0) 5 (13.9) Sometimes 5 (13.9) 10 (27.8) Rarely 2 (5.6) 7 (19.4) Never 0 (0.0) 8 (22.2) Do not remember 2 (5.6) 3 (8.3) 22. Would the child accept semi-solid, solid, and varied food in their diet? Always 12 (33.3) 18 (50.0) 0.198 Often 14 (38.9) 7 (19.4) Sometimes 8 (22.2) 6 (16.7) Rarely 2 (5.6) 3 (8.3) Never 0 (0.0) 2 (5.6) Do not remember 0 (0.0) 0 (0.0) 23. Would the mother alternate moments of dedication to the child with other interests? Always 11 (30.6) 6 (16.7) 0.564 Often 12 (33.3) 13 (36.1) Sometimes 8 (22.2) 9 (25.0) Rarely 3 (8.3) 6 (16.7) Never 1 (2.8) 2 (5.6) Do not remember 1 (2.8) 0 (0.0) 24. Would the child deal well with brief absences by the mother and react to long ones? Always 11 (30.6) 7 (19.4) 0.179 Often 11 (30.6) 9 (25.0) Sometimes 8 (22.2) 5 (13.9) Rarely 5 (13.9) 7 (19.4) Never 1 (2.8) 7 (19.4) Do not remember 0 (0.0) 1 (2.8) 25. Would the mother offer toys as an alternative to the interest of the child for the mother’s body? Always 11 (30.6) 3 (8.3) 0.122 Often 10 (27.8) 12 (33.3) Sometimes 8 (22.2) 7 (19.4) Rarely 2 (5.6) 3 (8.3) Never 4 (11.1) 5 (13.9) Do not remember 1 (2.8) 6 (16.7) 26. Would the mother no longer feel obliged to satisfy all the child would ask for? Always 4 (11.1) 8 (22.2) 0.037 Often 9 (25.0) 3 (8.3) Sometimes 15 (41.7) 7 (19.4) Rarely 4 (11.1) 7 (19.4) Never 3 (8.3) 10 (27.8) Do not remember 1 (2.8) 1 (2.8) 27. Would the child look curiously toward what was interesting to the mother? Continues... DISCUSSION In a study that examined the discriminative ability of the IRDl questionnaires among ASD, mental retardation, and nor- mality by administering the instrument to family videos of chil- dren who were later on diagnosed with autism, the author found compatible results when verifying that the average chance of an autistic baby to show like or dislike toward something is 1.82 times higher than that of a normal baby(21). The importance of using screening and tracking tools for the early diagnosis of ASD is widely discussed in the litera- ture(11-16,18,20-22). However, these instruments should have some basic features such as being easy to be applied, with simple questions and answers, being lowincost and being effective in the tracking which it is proposed to(14,19). Another study of family movies of children who were later on diagnosed with autism found that the social profile of an autistic baby differs from that of a normal baby when consid- ering the average duration of glances addressed to the parents and the duration and frequency of social smiling(26). The use of the IRDIquestionnaire, in the sample stud- ied, showed itself to be adequate in relation to the under- standing of the questions and the pattern of answers given by the interviewees, as well as being quick and easy to be applied. The IRDI questionnaire showed good internal consistency. This result is consistent with other reliability studies of instruments used for the screening and diagnosis of ASD: Autism Screening Questionnaire, CARS-BR (the Brazilian version of the Childhood Autism Rating Scale) and Childhood Autism Rating Scale(20,23,24). The literature is vast and consensual when pointing out the deficits in social skills and in shared attention as strong early signs of ASD(16,27). Thus, the studies corroborate the results obtained here by claiming that, in children with ASD, the lack of interest in social stimuli is characterized by reduced fre- quency of eye contact; these children having a preference for non-social stimuli, manifested by directing the gaze preferen- tially to objects and not to individuals, along with the lack of interest in sharing attention(16,27-29). The results show that many of the questions whose answers had statistical significance include important signs of risk for ASD, such as the exchange of looks, shared attention, and social interaction. An important concept introduced by Laznik et al.(30) regarding the three stages of the instinctive cycle is dis- cussed in Question 15 of the IRDIquestionnaire. RESULTS Always 12 (33.3) 3 (8.3) <0.001 Often 16 (44.4) 7 (19.4) Sometimes 8 (22.2) 10 (27.8) Rarely 0 (0.0) 9 (25.0) Never 0 (0.0) 5 (13.9) Do not remember 0 (0.0) 2 (5.6) CoDAS 2014;26(2):138-47 145 χ2 test Table 3. Continuation Variable Category Group p-value Control Study n % n % 28. Would the child like to play with objects used by the parents? Always 14 (38.9) 3 (8.3) 0.002 Often 13 (36.1) 10 (27.8) Sometimes 7 (19.4) 9 (25.0) Rarely 0 (0.0) 8 (22.2) Never 1 (2.8) 5 (13.9) Do not remember 1 (2.8) 1 (2.8) 29. Would the mother request the child to name what they wanted, not settling for gestures alone? Always 16 (44.4) 14 (38.9) 0.287 Often 9 (25.0) 16 (44.4) Sometimes 7 (19.4) 2 (5.6) Rarely 2 (5.6) 2 (5.6) Never 2 (5.6) 1 (2.8) Do not remember 0 (0.0) 1 (2.8) 30. Would the parents impose minor behavior rules to the child? Always 18 (50.0) 12 (33.3) 0.302 Often 12 (33.3) 16 (44.4) Sometimes 6 (16.7) 5 (13.9) Rarely 0 (0.0) 1 (2.8) Never Do not remember 0 0 (0.0) (0.0) 2 0 (5.6) (0.0) 31. Would the child differentiate their parents’ objects from their own? Always 9 (25.0) 3 (8.3) <0.001 Often 16 (44.4) 5 (13.9) Sometimes 9 (25.0) 9 (25.0) Rarely 0 (0.0) 11 (30.6) Never 0 (0.0) 6 (16.7) Do not remember 2 (5.6) 2 (5.6) Total 36 (100) 36 (100) *FPM and MCC were responsible for defining the design of the study; FPM performed the data collection and literature survey, which were analyzed and discussed together with MCC and RRRP. CONCLUSION CONCLUSION to seize it. The second stage, the reflective and self-erotic one, is the one in which the child takes the object as a part of their own body (e.g., thumb/hand sucking). The third stage is the one in which the child is offered as the object of another sub- ject (e.g., when the baby stretches their foot toward the mother’s mouth and the two of them experience a shared pleasure in this game). According to the author, the autistic child does not reach the third stage of the cycle(30). Without denying the effect of the organic factors involved in this clinical panorama, the failure in settling the third stage of the instinctive cycle suggests that the possibility of placing themselves in someone’s place, when the baby offers themselves as an object to others, does not occur in the case of autistic children(30). This research started from the finding that the delay in language development is one of the most common concerns of parents of chil- dren with ASD, which is why the Speech-Language Pathologist is, in many cases, the first professional sought by the family. Thus, the role of these professionals in identifying the potential risk for ASD and the importance of their conduct as to redirecting the patients toward relevant clinical care are evident. Aiming to support and endorse such actions, this research presented a proposal for the adapting of the IRDI into a retro- spective questionnaire for parents (IRDIquestionnaire). It was found that the questionnaire items covered the main early clinical signs reported in the literature. It was also notice- able that the answers by the parents have pointed out signifi- cant differences between the SG and the CG. The IRDIquestionnaire also contains some questions con- cerning the participation of the mother and her attitudes in rela- tion to their child’s behavior. The results of this study showed that two among these were noticeable after the statistical anal- ysis conducted. To that extent, worth noting is the peculiarity introduced by the research that has elaborated the IRDI, includ- ing the relationship between mother and baby to the center of observation, differently from what predominantly occurs when it comes to signs of risk for ASD, focusing their observation solely on the child(18,21,22). REFERENCES In the same vein, it is noteworthy that the issues that have the “motherese” theme have not been highlighted in the sta- tistical analysis performed in this study. The studies in psy- cholinguistics of the prosody of mothers of children with autism show significant differences in the melodic curves analyzed — a fact that certainly served as base for the inclu- sion of items that address this aspect in the preparation of the IRDI(30). However, in a study conducted with the application of the IRDl questionnaires to family videos, there were also no differences in the analysis of these items, which was attrib- uted, in the study, to the possibility that the ability to respond to the calling by others is not yet affected in the autistic group from 0 to 3 months of age(27). i 1. Volkmar FR, Pauls D. Autism. Lancet. 2003;362(9390):1133-41. 2. Thurm A, Lord C, Lee LC, Newschaffer C. Predictors of language acquisition in preschool children with autism spectrum disorders. J Autism Dev Disord. 2007;37:1721-34. 3. Eigsti IM, Bennetto L, Dadlani MB. Beyond pragmatics: morphosyntactic development in autism. J Autism Dev Disord. 2007;37:1007-23. 4. Weismer SE, Lord C, Esler A. Early language patterns of toddlers on the autism spectrum compared to toddlers with developmental delay. J Autism Dev Disord. 2010;40:1259-73. 5. Matson JL, Wilkins J, Gonzalez M. Early identification and diagnosis in autism spectrum disorders in young children and infants: how early is too early? Res Autism Spectr Disord. 2008;2:75-84. 6. Wetherby AM, Woods J, Allen L, Cleary J, Dickinson H, Lord C. Early indicators of autism spectrum disorders in the second year of life. J Autism Dev Disord. 2004;34(5):473-93. From the findings presented, it appears that much of the content of the IRDIquestionnaire is consistently referenced in the literature, emphasizing signs of significant risk for ASD. Thus, it is suggested that this instrument may support future researches on the subject, particularly as to the early diagnosis of ASD. 7. Dewrang P, Sandberg AD. Parental retrospective assessment of development and behavior in Asperger syndrome during the first 2 years of life. Res Autism Spectr Disord. 2010;4:461-73. 8. Horovitz M, Matson JL, Tuygin N, Beighley JS. The relationship between gender and age of first concern in toddlers with autism spectrum disorders. Res Autism Spectr Disord. 2012;6:466-71. This study has some limitations. CONCLUSION The application of the IRDIquestionnaire also included an important recommendation as to identification, tracking, and sorting tools: ease of application and low cost, enabling their use on a large scale. Other studies with the IRDIquestionnaire are recommended, aiming at the understanding of its scope and limitations. Therefore, it is suggested that studies with the IRDIquestionnaire should continue, so that it can be effectively validated and used as a tool for tracking ASD. The results obtained here differ from those of the multi- center study conducted with the IRDI, which showed a group of 15 indicators with the ability to predict the risk for child development, out of which only 5 would match those indicated in this study, according to the adopted criterion for statistical significance. This might be because the present study focused on children with ASD, unlike what happened with the research mentioned, which has focused its attention on the problems of child development more broadly. CoDAS 2014;26(2):138-47 DISCUSSION According to the authors, the subjective constitution is given in three stages: the first stage, called active, is the one in which the baby goes in search of the external object (oral) in order The matter of exchange of glances or looks between the child and the mother addresses a sign of ASD widely studied in the literature(1,5,13,14,25). 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https://openalex.org/W2609143063	https://bmcgenomics.biomedcentral.com/track/pdf/10.1186/s12864-017-3674-x	English	null	The predictive nature of transcript expression levels on protein expression in adult human brain	BMC genomics	2,017	cc-by	9,184	Bauernfeind and Babbitt BMC Genomics (2017) 18:322 DOI 10.1186/s12864-017-3674-x Bauernfeind and Babbitt BMC Genomics (2017) 18:322 DOI 10.1186/s12864-017-3674-x © 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 predictive nature of transcript expression levels on protein expression in adult human brain Amy L. Bauernfeind1,2* and Courtney C. Babbitt3* Amy L. Bauernfeind1,2* and Courtney C. Babbitt3* Abstract Background: Next generation sequencing methods are the gold standard for evaluating expression of the transcriptome. When determining the biological implications of such studies, the assumption is often made that transcript expression levels correspond to protein levels in a meaningful way. However, the strength of the overall correlation between transcript and protein expression is inconsistent, particularly in brain samples. Results: Following high-throughput transcriptomic (RNA-Seq) and proteomic (liquid chromatography coupled with tandem mass spectrometry) analyses of adult human brain samples, we compared the correlation in the expression of transcripts and proteins that support various biological processes, molecular functions, and that are located in different areas of the cell. Although most categories of transcripts have extremely weak predictive value for the expression of their associated proteins (R2 values of < 10%), transcripts coding for protein kinases and membrane- associated proteins, including those that are part of receptors or ion transporters, are among those that are most predictive of downstream protein expression levels. Conclusions: The predictive value of transcript expression for corresponding proteins is variable in human brain samples, reflecting the complex regulation of protein expression. However, we found that transcriptomic analyses are appropriate for assessing the expression levels of certain classes of proteins, including those that modify proteins, such as kinases and phosphatases, regulate metabolic and synaptic activity, or are associated with a cellular membrane. These findings can be used to guide the interpretation of gene expression results from primate brain samples. Keywords: RNA-Seq, Gene, Proteomics, Chimpanzee Keywords: RNA-Seq, Gene, Proteomics, Chimpanzee * Correspondence: amybauernfeind@wustl.edu; cbabbitt@bio.umass.edu 1Department of Neuroscience, Washington University Medical School, St. Louis, MO 63110, USA 3Department of Biology, University of Massachusetts Amherst, Amherst, MA 01003, USA Full list of author information is available at the end of the article Results Overall, we report R2 values of 0.07 in the expression levels of 815 transcript/protein pairs in the human ACC. This result is higher than we had reported previously [6] because of the inclusion of more transcript/protein pairs in this expanded dataset that is not limited to homolo- gous proteins between humans and chimpanzees. Not- ably, the coefficient of determination is similar to that reported by Wei and colleagues in adult human and Rhesus macaque brain tissue [12]. We performed ordin- ary least squares (OLS) regressions on the transcript and protein expression levels within each GO category of biological process, molecular function, and cellular com- ponent. In human ACC, 306 categories of biological pro- cesses, 125 categories of molecular functions, and 104 categories of cellular components were represented by 10 or more transcript/protein pairs. A complete ordered list of the transcript and protein expression levels in hu- man ACC and the OLS regression results including the predictive natures (R2) of GO categories can be found in Additional file 1. When appropriate, transcripts are asso- ciated with more than one category, which accounts for the similarities between functional groupings (eg. “or- ganelle outer membrane” and “outer membrane”). In the spirit of exploring the correlation between tran- script and protein expression that should be expected from non-model samples, our objective was to deter- mine how transcript expression predicts protein expres- sion within molecular categories that are specific to brain tissue (eg. ‘synapse’) and with limitations that are common in many studies of molecular expression (ie. PMIs of up to 8 h and heterogenous cell populations). We predict that molecules within the same Gene Ontol- ogy (GO) attributes (i.e. biological process, molecular function, or cellular component) may share regulatory mechanisms associated with synthesis and degradation, which may affect the degree of correlation between tran- script and protein expression levels [10, 11]. Here, we ask whether there are certain classes of transcripts that are more predictive of protein expression levels than others by using transcriptomic and proteomic expression from our previously published dataset [6]. Specifically, we examined expression levels of transcripts and pro- teins by GO category to determine if different predictive relationships (coefficients of determination, R2 values) exist between molecules that participate in certain func- tions or are located in certain parts of the cell. Are there molecular attributes that suggest a stronger predictive relationship between transcripts and proteins than others? Background aspects of molecular phenotype has yet to be fully understood. In fact, the correlation between transcript expression levels and their protein products have gener- ally been found to be quite low and may vary across tis- sues and cell types [1–5], calling into question what biological significance can be drawn from transcriptomic and proteomic results. Next generation sequencing, including RNA-Seq, allows researchers to investigate transcript expression using label-free technology, and its relative ease of use has made this method the dominant technology for assessing molecular phenotype. When interpreting transcriptomic results, the assumption is frequently made that the ex- pression level of a transcript reflects that of the down- stream protein, suggesting the equivalence of these two molecules. However, the relationship between these two Our earlier study [6] explored transcript (RNA-Seq) and protein (liquid chromatography with tandem mass spectrometry [LC/MS/MS]), expression in the anterior cingulate cortex (ACC) and caudate nucleus (CN) of humans and chimpanzees in order to determine if differ- ential expression analyses of these two molecules re- sulted in different interspecific biological signals. Importantly, we reported that both species display a lower degree of correlation between transcript and Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 2 of 11 Page 2 of 11 protein expression levels (human R2 = 0.03; chimpanzee R2 = 0.04) than typically reported in other organisms and tissues [1, 2]. The correlation between transcript and protein expression in mammalian cells is generally mod- est, ranging from 9% in human monocytes to 40% in mouse fibroblasts [7, 8], but these and other inquiries into the relationship between transcript and protein ex- pression levels have done so using homogenous cell cul- tures in an effort to limit confounding variables [9]. Many transcriptomic studies, however, must overcome additional challenges imposed by longer postmortem in- tervals (PMIs) and greater cellular heterogeneity than these carefully controlled studies. cellular locations, and we explore possible causes of this variation by investigating correlations between categor- ical R2 value and category size, molecular abundance, gene length, and previously published rates of molecular synthesis and degradation [8]. Understanding implicit biases in transcriptomic and proteomic data is funda- mental to answering questions pertaining to the molecu- lar phenotype of the brain or any other biological tissue. Results Strong predictive relationships suggest that the results of RNA-Seq would be informative of expression levels of downstream proteins, while the expression levels of classes of transcripts with weaker predictive re- lationships offer little value in predicting downstream protein abundance. g Descriptive statistics were performed to summarize the R2 values across GO categories that contained a minimum of 10 transcript/protein pairs. Because the analyses were dependent upon the categories included and some tran- script/protein pairs were represented in more than one category, we used the descriptive statistics as a way of de- scribing what R2 results may be expected from specific GO categories, while acknowledging these limitations. Categories that contained the same molecules and had identical R2 values as another category, including 45 categories of biological process, 10 categories of molecular function, and 10 categories of cellular component, were deleted from the dataset. Categories of biological process yielded R2 values between < 0.01 to 0.51 (mean = 0.15 ± 0.11, median = 0.12). For molecular function, categories pro- duced R2 values that ranged from < 0.01 to 0.47 (mean = 0.14 ± 0.10, median = 0.11). Categories of cellular compo- nent yielded R2 values between < 0.01 to 0.66 (mean = 0.12 ± 0.11, median = 0.08). The central tendency of R2 values for categories of biological process was statistically higher than that of cellular components (Mann Whitney In general, we found that most transcripts have fairly low predictive value for determining protein expression levels, falling within one standard deviation of the mean for randomly associated transcript/protein pairs. How- ever, we found that transcripts that coded for membrane-bound proteins, in particular those that have oxidoreductase and synaptic functions, and protein ki- nases and phosphatases were most predictive of protein expression. Our results indicate that the predictive value of transcripts is not uniform across all functions or Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 3 of 11 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 U = 13,941, p < 0.05), but comparisons revealed that those of the other annotations were statistically equivalent. Permutation tests We performed permutation tests in which random tran- scripts and proteins were classified into categories to mimic our observed data. The category sizes were sam- pled and replaced from the actual sizes of our observed categories for molecular function and cellular compo- nent to ensure that the range of possible category sizes represented our dataset. The resampling occurred over 1000 iterations. In the permutation test to mimic the an- notation of biological process, randomly paired tran- scripts and proteins yielded R2 values that ranged from < 0.01 to 0.60 (mean = 0.05 ± 0.08, median = 0.02). As ex- pected, the central tendency of the observed data was higher than that of the permutation test (Mann Whitney U = 207,103, p < 0.001). For molecular function, categor- ies of random transcripts and proteins produced R2 values that ranged from < 0.01 to 0.65 (mean = 0.05 ± 0.07, median = 0.02). The R2 values for molecular func- tion permutation test compared to the observed data were inequivalent (Mann Whitney U = 94,099, p < 0.001) with the median of the observed data greater than that of the permutation test. The permutation test of cellular component categories yielded R2 values between < 0.01 and 0.64 (mean = 0.05 ± 0.08, median = 0.01). For cellular component, the central tendency of the observed R2 values was higher than that of the permutation test (Mann Whitney U = 74,470, p < 0.001). We listed the biological processes, molecular func- tions, and cellular locations of transcripts that were most predictive of the expression levels of their associated proteins (Table 1). Proteins involved in the addition (ki- nases) or removal (phosphatases) of a phosphate group and transmembrane proteins, including those that are components of receptors or ion channels and have those that have oxidoreductase functions, are among the mole- cules that display the highest correlation in their expres- sion to their parent transcript (Fig. 2). However, the slopes display a fairly wide range of confidence intervals, reflecting the fact that the relationship between tran- script and protein expression can be quite variable within a GO category. Permutation tests The interpretation of our results rely on coefficients of determination, R2 values, which are a similar measure of how closely the data are fitted to the regression line, and we interpret this value as a measure of how predictable protein expression levels are from the expression levels of their parent transcripts. Like the observed data, the distributions of the R2 values for all three permutation tests deviated from normality with a rightward skew (biological process Shapiro-Wilk = 0.67, p < 0.001, skewness = 2.63; molecular function Shapiro-Wilk = 0.63, p < 0.001, skewness = 3.19; cellular component Shapiro-Wilk = 0.62, p < 0.001, skewness = 2.76). As expected, the permutation tests revealed strong 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 5 10 15 20 Cumulative Percentage R2 Fig. 1 Cumulative percentages of R2 values. GO categories of biological process (green), molecular function (red) and cellular component (blue) are plotted together. The lighter green, red, and blue lines represent the results of permutation tests that produced 1000 OLS regressions from randomly sampled transcripts and proteins of equivalent categorical sizes of biological process, molecular function, and cellular component, respectively. The observed data from all three GO annotations have distributions with a positive skew, displaying how there are categories of both molecular function and cellular components that are more predictive of protein expression than randomly associated transcripts and proteins. The data is plotted with a bin width is 0.05, and the representative line is Gaussian smoothed. Duplicate categories that contain the same molecules and have the same R2 value as another category are not represented 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0 5 10 15 20 Cumulative Percentage R2 Fig. 1 Cumulative percentages of R2 values. GO categories of biological process (green), molecular function (red) and cellular component (blue) are plotted together. The lighter green, red, and blue lines represent the results of permutation tests that produced 1000 OLS regressions from randomly sampled transcripts and proteins of equivalent categorical sizes of biological process, molecular function, and cellular component, respectively. The observed data from all three GO annotations have distributions with a positive skew, displaying how there are categories of both molecular function and cellular components that are more predictive of protein expression than randomly associated transcripts and proteins. The data is plotted with a bin width is 0.05, and the representative line is Gaussian smoothed. Results For each GO annotation, the distributions of categories had positive skews (biological process Shapiro-Wilk = 0.92, p < 0.001, skewness = 0.98; molecular function Shapiro- Wilk = 0.95, p < 0.001, skewness = 0.71; cellular compart- ment Shapiro-Wilk = 0.83, p < 0.001, skewness = 1.87, re- spectively), suggesting that while most R2 values fall near the median for the annotation other categories have much higher R2 values. These categories contain transcripts with greater predictive value. Interestingly, cellular component revealed the largest positive skew, a result largely driven by the highly predictive relationship between transcripts and proteins associated with the cellular ‘outer membrane’ (R2 = 0.66) (Fig. 1). Correlation with category size Correlation with category size 0.0 0.2 0.4 0.6 Category size 200 400 600 0.0 0.2 0.4 0.6 0.0 0.2 0.4 0.6 Category size 200 400 600 Biological Process Molecular Function Cellular Component Category size 200 400 600 regulation of protein modification process positive regulation of cell communication phosphatase activity outer membrane membrane lipid metabolic process regulation of phosphate metabolic process regulation of synaptic plasticity regulation of kinase activity protein amino acid phosphorylation phosphate metabolic process cellular protein complex assembly phosphorylation endocytosis calcium ion transport membrane coat extracellular matrix organelle lumen mitochondrial membrane part protein kinase activity protein domain specific binding phosphotransferase activity alcohol group as acceptor manganese ion binding ATPase activity coupled to transmembrane movement of ions phosphoprotein phosphatase activity R2 R2 R2 Fig. 2 Coefficients of determination for GO categories of molecular function and cellular component. R2 values are typically quite low but certain categories (labeled) display greater predictive value than others. These categories have R2 values that exceed the mean of the 1000 resampled categories by approximately four standard deviations (two standard deviations above the means for the respective annotations) We were interested as to whether GO category size had an affect on the overall predictive nature of transcripts and proteins within a given annotation of our observed data. We found Spearman’s correlation coefficients be- tween the categorical R2 value and the number of genes per GO category in our dataset. The overall correlation between R2 value and number of genes was negative for biological process, molecular function, and cellular com- ponent (Table 2), however this relationship was only sig- nificant for cellular compartment. The strongly negative relationship between R2 value and category size in cellu- lar compartment may be due to an increase in diversity in functional processes represented within a category as the categorical size increases. The same effect was ob- served under the annotation of molecular function when categories were limited to those with more than 20 tran- script/protein pairs. By definition, the GO annotation of “biological process” must have more than one distinct step [13] and therefore, the annotation contains genes with a greater diversity compared to the molecular func- tion annotation regardless of the size of the category. This observation may account for the fact that no correl- ation is observed between R2 value and category size within the annotation of biological functions. Correlation with molecular abundance Because low molecular expression may affect the accur- acy of abundance estimates, we examined the correlation between average transcript and protein abundances and the R2 values for individual GO categories. We found no significant correlations between mean gene or protein expression and categorical R2 value, indicating molecular abundance has no bearing on the predictability of the transcript/protein relationship (Table 2). Correlation with gene length Fig. 2 Coefficients of determination for GO categories of molecular function and cellular component. R2 values are typically quite low but certain categories (labeled) display greater predictive value than others. These categories have R2 values that exceed the mean of the 1000 resampled categories by approximately four standard deviations (two standard deviations above the means for the respective annotations) Spearman correlation coefficients were found between gene length and R2 value. Biological process, molecular function, and cellular component categories did not pro- duce significant relationships between these measures (Table 2). Permutation tests Duplicate categories that contain the same molecules and have the same R2 value as another category are not represented Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 4 of 11 Table 1 GO categories with the highest predictive value between transcript/protein pairs Category n R2 p Slope Slope CI Intercept Intercept CI Biological process regulation of protein modification process 10 0.51 0.0211 0.38 0.07 – 0.68 4.06 3.37 – 4.75 positive regulation of cell communication 11 0.50 0.0145 0.47 0.12 – 0.82 3.86 3.05 – 4.68 membrane lipid metabolic process 11 0.47 0.0192 0.75 0.15 – 1.35 2.99 1.52 – 4.46 regulation of phosphate metabolic process 10 0.47 0.0290 0.38 0.05 – 0.7 4.08 3.34 – 4.82 regulation of synaptic plasticity 10 0.46 0.0317 0.73 0.08 – 1.38 3.58 1.88 – 5.29 regulation of kinase activity 15 0.44 0.0070 0.76 0.25 – 1.27 3.16 1.85 – 4.46 protein amino acid phosphorylation 34 0.43 0.0000 0.56 0.33 – 0.79 3.8 3.27 – 4.33 phosphate metabolic process 55 0.40 0.0000 0.57 0.38 – 0.76 3.75 3.31 – 4.19 cellular protein complex assembly 16 0.40 0.0089 0.67 0.2 – 1.15 4.02 2.87 – 5.17 phosphorylation 41 0.38 0.0000 0.56 0.33 – 0.79 3.82 3.3 – 4.35 endocytosis 19 0.37 0.0055 0.57 0.19 – 0.95 4.07 3.2 – 4.93 calcium ion transport 11 0.37 0.0483 0.65 0.01 – 1.3 3.72 2.04 – 5.39 DNA metabolic process 11 0.35 0.0548 0.6 -0.02 – 1.21 3.77 2.48 – 5.05 negative regulation of protein metabolic process 19 0.35 0.0081 0.45 0.13 – 0.76 4.08 3.23 – 4.92 cell cycle 14 0.35 0.0272 0.5 0.07 – 0.93 3.7 2.77 – 4.62 positive regulation of cell proliferation 20 0.34 0.0068 0.41 0.13 – 0.69 4.02 3.50 – 4.54 regulation of hydrolase activity 18 0.34 0.0110 0.53 0.14 – 0.92 3.91 2.98 – 4.84 regulation of actin filament length 12 0.34 0.0477 0.48 0.01 – 0.96 4.18 2.87 – 5.49 cell projection organization 14 0.33 0.0306 0.67 0.07 – 1.26 3.64 2.00 – 5.28 regulation of growth 12 0.33 0.0499 0.51 0 – 1.02 3.93 2.67 – 5.18 regulation of cell growth 10 0.33 0.0824 0.48 -0.08 – 1.04 3.89 2.55 – 5.24 positive regulation of cellular process 64 0.31 0.0000 0.44 0.27 – 0.61 4.16 3.77 – 4.54 protein modification process 65 0.30 0.0000 0.46 0.28 – 0.64 3.96 3.55 – 4.38 Molecular function phosphatase activity 16 0.47 0.0036 0.97 0.37 – 1.56 2.67 1.24 – 4.11 phosphoprotein phosphatase activity 12 0.41 0.0241 0.72 0.11 – 1.32 3.29 1.83 – 4.74 protein kinase activity 33 0.37 0.0002 0.55 0.28 – 0.81 3.76 3.2 – 4.33 protein domain specific binding 18 0.37 0.0079 0.55 0.16 – 0.93 3.74 2.82 – 4.65 phosphotransferase activity alcohol group as acceptor 45 0.35 0.0000 0.56 0.33 – 0.79 3.8 3.31 – 4.3 manganese ion binding 12 0.34 0.0451 -0.39 -0.76 – - 0.01 5.25 4.52 – 5.98 ATPase activity coupled to transmembrane movement of ions phosphorylative mechanism 17 0.34 0.0135 0.39 0.09 – 0.69 4.74 4.04 – 5.44 GTPase activity 52 0.33 0.0000 0.48 0.29 – 0.68 4.28 3.8 – 4.75 calmodulin binding 31 0.33 0.0008 0.52 0.24 – 0.81 4.11 3.4 – 4.83 ligase activity 20 0.31 0.0102 0.42 0.11 – 0.72 3.85 3.11 – 4.58 Cellular component outer membrane 10 0.66 0.0043 0.88 0.37 – 1.40 3.4 2.30 – 4.51 membrane coat 13 0.46 0.0107 0.76 0.21 – 1.30 3.58 2.25 – 4.90 extracellular matrix 11 0.38 0.0435 0.67 0.02 – 1.32 3.71 2.16 – 5.25 organelle lumen 10 0.37 0.0612 -0.38 -0.79 – 0.02 5.93 4.98 – 6.89 mitochondrial membrane part 21 0.36 0.0043 0.84 0.30 – 1.39 3.23 1.94 – 4.52 cell surface 12 0.30 0.0630 0.35 -0.02 – 0.72 4.35 3.43 – 5.27 membrane-enclosed lumen 12 0.30 0.0638 -0.37 -0.77 – 0.03 6.01 5.07 – 6.94 Data in bold are the subcategories Page 5 of 11 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Production and degradation rates error t-value p Biological process Intercept 4.23E-02 8.07E-02 0.52 0.60 Transcription rate 1.09E-02 4.06E-03 2.69 <0.01 Translation rate 8.86E-05 5.11E-05 1.73 0.08 mRNA half-life -4.32E-03 5.60E-03 −0.77 0.44 Protein half-life 5.61E-04 1.97E-04 2.84 <0.01 R2 = 0.08, F(4, 207) = 4.315, p-value = 0.002 Molecular function Intercept 3.41E-01 1.02E-01 3.35 <0.01 Transcription rate -8.63E-03 5.74E-03 −1.5 0.14 Translation rate 4.12E-05 2.89E-05 1.43 0.16 mRNA half-life -1.38E-02 6.71E-03 −2.06 <0.05 Protein half-life -2.58E-04 1.97E-04 −1.31 0.19 R2 = 0.13, F(4, 87) = 3.125, p-value = 0.02 Cellular compartment Intercept -1.89E-01 1.05E-01 -1.80 0.08 Transcription rate -2.99E-02 8.26E-03 −3.62 <0.01 Translation rate 8.71E-05 6.15E-05 1.42 0.16 mRNA half-life 3.63E-02 6.71E-03 5.40 <0.01 Protein half-life -8.21E-04 2.79E-04 −2.94 <0.01 R2 = 0.40, F(4, 63) = 10.41, p-value < 0.001 Data in bold are the subcategories Production and degradation rates The of the categorical regression lines were different between species, and the ranked correlation coefficients between humans and chimpanzees were similar (biological process in ACC: Spearman’s ρ = 0.79, p < 0.001; CN: ρ = 0.61, p < 0.001; molecular function in ACC: ρ = 0.82, p < 0.001; CN: ρ = 0.71, p < 0.001; cellular component in ACC: ρ = 0.77, p < 0.001; CN: ρ = 0.60, p < 0.001). The category as previously mentioned. The correlation be- tween R2 value and transcription rate is strongly negative for both molecular function and cellular compartment, indicating that transcripts with high rates of synthesis do not have strong predictive value for downstream protein abundance. Multiple regression analyses were performed to deter- mine how R2 values were related to four variables: rates of transcript and protein synthesis and their respective degradation rates. Table 3 summarizes the results. In the multiple regressions associated with all three GO anno- tations, translation rate is observed to have a positive weight on R2 value when controlling for other variables. However, this relationship is not significant for any GO annotation. For both molecular function and cellular compartment, mRNA half-life has a larger positive weight than translation rate and is significant for both annotations. Table 3 Results of multiple regression analyses of R2 value (dependent variable) against transcription and translation rates and mRNA and protein half-lives (independent variables) Table 3 Results of multiple regression analyses of R2 value (dependent variable) against transcription and translation rates and mRNA and protein half-lives (independent variables) Table 3 Results of multiple regression analyses of R2 value (dependent variable) against transcription and translation rates and mRNA and protein half-lives (independent variables) Estimate Std. Production and degradation rates Published mRNA and protein molecular half-lives and transcription and translation rates for mouse fibroblasts [8] allowed us to assess whether our observed R2 values were due to known differences in the molecular stability of mammalian cells. We observed strong correlations between R2 values and mean transcription rates, transla- tion rates, and mRNA half-lives, respectively for categor- ies of molecular function and cellular compartment. The same trend was not observed for biological processes, likely due to the diversity of functions included in the negative correlations between R2 values and category sizes (biological process Spearman’s ρ = -0.48, p < 0.001; mo- lecular function Spearman’s ρ = -0.41, p < 0.001; cellular component Spearman’s ρ = -0.55, p < 0.001), indicating that the inclusion of more randomly associated transcripts and proteins has a negative impact on the predictive rela- tionship of transcript and protein expression within a category. Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 6 of 11 Table 2 Spearman rank correlation coefficients (ρ) between the transcript/protein categorical R2 values and possible sources of variation Biological Process Molecular Function Cellular Compartment Category Size All categories −0.08 −0.05 −0.25** Categories with < 20 transcript/protein pairs −0.05 −0.05 −0.26 Categories with ≥20 transcript/protein pairs 0.03 −0.22* −0.19 Abundance Mean gene expression −0.09 −0.17 0.10 Mean protein expression −0.07 0.15 0.04 Gene Length All categories 0.05 −0.03 0.11 Synthesis/degradation Transcription rate 0.17* −0.31** −0.29* Translation rate 0.13 0.29 −0.30 mRNA half-life −0.10 −0.36** 0.26* Protein half-life 0.12 0.05 −0.24 p value of < 0.05 p value of < 0.01 Table 2 Spearman rank correlation coefficients (ρ) between the transcript/protein categorical R2 values and possible sources of of the categorical regression lines were different between species, and the ranked correlation coefficients between humans and chimpanzees were similar (biological process in ACC: Spearman’s ρ = 0.79, p < 0.001; CN: ρ = 0.61, p < 0.001; molecular function in ACC: ρ = 0.82, p < 0.001; CN: ρ = 0.71, p < 0.001; cellular component in ACC: ρ = 0.77, p < 0.001; CN: ρ = 0.60, p < 0.001). Interspecific and interregional comparisons We previously reported the expression levels of tran- script/protein pairs that are expressed in both humans and chimpanzees [6]. We used this previously published dataset of homologous transcript/protein pairs to deter- mine whether the predictive relationship of transcripts and proteins is similar between the two species and in two different regions of the brain. Using well- represented GO categories (≥10 transcript/protein pairs) in the ACC, we found 215 annotations representing biological processes, 100 for molecular functions, and 78 for cellular components. For CN, 195, 90, and 69 anno- tations were found for biological processes, molecular functions, and cellular components, respectively. The confidence interval for each OLS slope was compared between humans and chimpanzees to determine if the relationship between average transcript and protein expression differed between species. We found that none Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 7 of 11 Page 7 of 11 predictive relationships between transcripts and proteins were fairly similar in both ACC and CN (biological process: ρ = 0.43, p < 0.001; molecular function: ρ = 0.43, p < 0.001; cellular component: ρ = 0.75, p < 0.001). accounting for a maximum of 66% of the variation observed in mean categorical protein abundance. Most categories are not better or are not significantly different from random in their predictive values. p We explored possible sources of variability in R2 value by examining its correlation with category size, average transcript and protein expression, and gene length. We had predicted that hydrolases and other enzymes, which are typically short in length, may be particularly poor predictors of protein abundance due to their relatively fast RNA degradation [15, 16]. It is well understood that it is biologically beneficial for molecules with such func- tion to have carefully regulated half-lives [8, 17]. More- over, short proteins are notoriously problematic to quantify [18], making it difficult to assess whether this finding is due to technological limitations or true bio- logical differences. We report no correlation with regard to average expression levels or gene length at the level of GO category, but these factors may contribute to variation at the level of the individual transcript/protein pair. How- ever, the number of transcript/protein pairs within a GO category likely has an affect on the R2 value as evidence by the differing correlations that are apparent when GO an- notations of varying sizes are considered. Discussion In an earlier manuscript, we reported a very low correl- ation between transcript and protein expression in two brain regions, ACC and CN, of both humans and chim- panzees [6]. Coefficients of determination (R2) were roughly 0.03 for both species and brain regions, indicat- ing that transcript expression predicts 3% protein ex- pression. The current study extends those initial findings by asking whether grouping transcript/protein pairs by similar attributes produces better predictive outcomes. Although we found a higher overall R2 value in the current study (0.07), this result must be due to the greater number of transcript/protein pairs included in the dataset. Previously, other authors have theorized that transcripts/ proteins that contribute to a cell’s structure may offer a higher predictive value than those that are functionally modulated [7]. Similarly, neuronal compartmentalization may impose region-specific rates of translation in dispar- ate areas of the cell [14]. Both of these hypotheses would favor higher predictive values across specific transcript/ protein pairs compared to others as categorized by GO annotations. p The discordance between transcript and protein ex- pression levels may be caused by differential rates of transcription and translation and/or in vivo and post- mortem degradation rates of transcripts and proteins [8, 19–22]. While the stability of transcripts and proteins vary according to their functional characteristics [8, 23], the rate of translation has been found to be the most im- portant factor in predicting protein expression [24]. At the categorical level, translation rate weighs only slightly positively on the predictive value of transcript to protein abundance. Because mean molecular synthesis and deg- radation rates influence the R2 value but not in a con- sistent manner across GO annotations. it is likely that when considering transcripts and proteins by GO cat- egories these features (synthesis and degradation rates) that may otherwise help to explain molecular abundance do not have sufficient resolution. Other attributes, such as sequence features, may account for differences in pre- dictive value [25], which are not possible to account for at the categorical level. We report a large degree of diversity in R2 values when transcripts/proteins are categorized by their function or location in the cell. We find several trends in assessing the R2 values of transcript and protein abundance across GO categories. Interspecific and interregional comparisons Specifically, the inclusion of more transcript/protein pairs may have a negative effect on the overall predictive nature of the GO category since larger categories are by their very nature less specific than small categories (ie. between 10 and 20 transcript/protein pairs). Due to the negative association between R2 value and category size among the largest groupings of transcripts/proteins by function and location, it may be concluded that smaller, more specific categories of transcripts may be the most predictive of their asso- ciated proteins. We found the absolute value of the change in rank order of the R2 values of GO categories between humans and chimpanzees and between regions of the brain (Additional file 1). These scores represent differing rela- tionships between transcripts and proteins and higher values would suggest different regulatory measures act- ing on molecular expression [7, 14]. It is noteworthy that “cell death” and “nervous system development” are among those categories with the greatest change in R2 rank order between humans and chimpanzees. We note that categories such as “synapse” and “integral to plasma membrane” and those listed in Table 1 as having particu- larly high R2 values, display concordant R2 values across species and regions of the brain. Discussion First, perhaps by nature of the diverse func- tions and locations of the molecules contained within the categories, the annotation of biological process contains a large range of predictive values that cannot be explained by category size, molecular abundance, or molecular stability. This result may be the result of the large diversity of functions represented within each category of biological process. Under any annotation, the expression levels of transcripts grouped by GO category are capable of Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 8 of 11 Page 8 of 11 Despite the fact that the predictive values of many cat- egories of transcripts and proteins are no better than ran- dom, we wish to emphasize that several categories have very high R2 values considering the complex dynamics of transcription rates and molecular degradation. We report that transcripts coding for protein kinases, phosphatases, and membrane-associated proteins, especially those that participate in metabolic oxidoreductase activity or the transport of ions, are among the transcripts that are most predictive of their downstream protein expression levels. It is noteworthy that many of these categories of proteins are critical for aerobic metabolism. In short, molecules supporting these specific processes may have a better cor- relation between their transcripts and protein expression levels than other categories. decoupling of transcript and protein expression in- creases with age and may suggest an accretion of age- dependent post-translational regulation in primate brain [12]. Like the current study, concordance was found in transcript and protein expression levels within categories enriched for nucleotide and ATP binding. In the path- ways that displayed an age-related discordance in tran- script and protein levels, Wei et al. [12] found that regulatory and signaling functions were enriched for mTOR signaling and metabolic functions. The authors suggest that the increasing discordance between tran- script and protein expression as the lifespan progresses may be the result of mRNA binding proteins or other regulatory factors and contribute to aging and perhaps Alzheimer’s disease. Comparing our results to that of other studies suggests differences in the predictive value of genes across tissues. Guo and colleagues [7] found the predictive value of mRNA to protein expression in human monocytes, which were chosen for their relative homogeneity across cell types. The authors found transcript expression was poorly predictive of protein expression (R2 = 0.09), a re- sult similar to our own. Conclusions In the ACC and CN of the human and chimpanzee brain, we observe that the predictive nature of proteins can range from no predictive value whatsoever to fairly high. We find that transcripts that code for proteins that are integral to the membrane and support protein kinase and oxidoreductase activity are more predictive of pro- tein expression than the vast majority of other categor- ies. We conclude that it is important to consider the predictive nature of transcript/protein pairs when deter- mining the functional implications of gene expression studies. It may be practical to consider transcript and protein expression as two separate aspects of molecular phenotype, each with its own contribution to biological function. In the future, the challenge for molecular ex- pression studies will be to integrate transcript and pro- tein biology into a single unified message of tissue and cell function. We report our findings from fresh, frozen human brain tissue with PMIs of less than 8 h. It is not known how much stronger the relationship would be in fresh brain tissue. Rather than affecting all tran- scripts equally, postmortem degradation appears to target different classes of transcripts at varying rates [15]. Specifically, longer coding regions and 3’ UTRs cor- relate with more rapid degradation than the rest of the transcriptome [15, 23, 27, 28]. The most severe postmor- tem degradation occurs after 8 h and would not be a fac- tor in our study [15]. It is not known how postmortem degradation affects proteins of different functional classes. Discussion However, they report the highest correspondence between transcript/protein pairs in the extracellular region, whereas we found that those mole- cules that were intrinsic to the cellular membrane dis- played the strongest correlation in expression. The extracellular categories in our dataset (“extracellular re- gion” and “extracellular space”) are among the weaker transcript/protein correlations in the brain. Comparing the data from Guo and colleagues [7] with the current study suggests that there is a strong tissue-specific com- ponent to the relationship between transcripts and pro- teins. Furthermore, future work may reveal that the vast heterogeneity in neuronal transcript expression [24, 26] may also affect cellular-specific protein expression and may have profound implications for neuronal function. In general, our results were similar between species (humans and chimpanzees) and regions of the brain. It is likely that the predictive relationships, particularly in the categories with the highest R2 values, outlined here would hold true for other brain tissue in primates and perhaps mammals as a whole. We focused our attention on results from ACC in this study, but we note that our findings in CN were very similar, suggesting that the ability to predict protein expression levels from tran- script expression probably do not change across different regions of the brain. Additionally, it is worth noting that the results of this study are category-specific and that the correlation of individual transcript/protein pairs con- tained within a category can vary. Methods Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders at the University of Maryland (Baltimore, MD) and were free from neurological disorders. Frozen brain samples from adult common chimpanzees, Pan troglodytes (aged 23 to 35 years), were obtained from the National Chim- panzee Brain Resource (Washington, DC). The chim- panzees had been cared for according to Federal and Institutional Animal Care and Use guidelines and died of natural causes. All brain tissue was collected with a postmortem interval of less than 8 h and stored at -80 ° C. p g We were interested in determining whether category size, abundance levels, or gene length had an affect on the R2 levels produced by GO categories. We found Spearman correlation coefficients between category size (number of transcript/protein pairs within our dataset) and R2 value across GO annotations of biological process, molecular function, and cellular component. Next, we found mean abundance levels of transcripts and proteins and examined whether a correlation existed between these values and the R2. Finally, we found gene lengths by searching the RefSeq annotations for the lat- est human genome build, hg38, from the University of California Santa Cruz Table Browser [37]. Average gene length per GO category was compared to R2 value to de- termine if length affected transcript/protein predictive value. postmortem interval of less than 8 h and stored at 80 C. Brain tissue was sampled from the ACC and CN from adult humans (n = 3) and chimpanzees (n = 3). Each sample was divided into two pieces: one for RNA- sequencing and one for quantitative proteomics. Librar- ies were constructed from poly-A-enriched RNA of 30 million 50-bp sequences. Orthologous gene models were constructed for each species, and sequences were mapped to species-specific genomes (hg19 and panTro3) [29, 30]. For the majority of the analyses, a dataset from human ACC was used which contained 815 transcript/ protein pairs. However, to assess the variation observed between species and brain regions the expression levels of 522 transcript/protein pairs from human and chim- panzee ACC and 499 from human and chimpanzee CN were analyzed. Both transcriptomic and proteomic data were normalized in edgeR [31]. We combined our transcript and protein expression data with the molecular stability measures reported by Schwanhäusser and colleagues [8]. Methods Specifically, the au- thors had found transcription (molecules/[cellh]) and translation (molecules/[mRNA*h]) rates in addition to mRNA and protein half-life time (h). The merged data- set contained molecular abundance and stability mea- sures from 471 transcript/protein pairs. Consequently, fewer GO categories contained 10 or more transcript/ protein pairs (biological process: 212 categories; molecu- lar function: 92; cellular compartment: 68). Spearman rank correlation coefficients were found between the synthesis and degradation rates and mean categorical R2 value. A multiple regression was performed for each gene annotation using the R2 value as the dependent and the synthesis and degradation rates as independent variables. To compare R2 values between regions of the brain and between species, we performed Spearman rank cor- relations of the categories in each of the three GO anno- tations. In order to assess which categories represented the largest change in rank order, we found the absolute value of the difference in rank order position. These values were then scaled by dividing by the number of categories in the annotation and multiplying by 100. In the current study, we assigned each transcript/ protein pair to their GO categories for the annota- tions of biological process, molecular function, and cellular component. Categories contained a minimum of 10 transcript/protein pairs. We used the species mean log-transformed expression data to perform a series of linear regressions for each category. We performed OLS linear regressions rather than using the reduced major axis (RMA) method. Although RMA regressions attempt to diminish variance along x- and y-axes [32], some authors have reported that RMA can decreases variation along the x-axis that may be biologically meaningful [33, 34]. OLS only accounts for error along the y-axis [35]. We focus our report on the coefficient of determination, R2 values, produced from OLS regressions of each GO category individually. All statistics were performed in R (version 3.1.3) [36], and the linear regressions were performed in the SMATR package (version 3.4). Additional file Additional file 1: Complete list gene and protein expression in human ACC and the results of the OLS regressions. The file also contains the rank order changes of categories when R2 values are compared between species or regions of the brain. (XLSX 9357 kb) Methods Although the current work only considers adult tissue, it is important to comment on the possibility of the rela- tionship between transcripts and proteins changing throughout the lifespan. A recent study in prefrontal cortex of humans and Rhesus macaques found that the This study used transcriptomic and proteomic expres- sion data that are available through our previously pub- lished work [6]. Briefly, frozen human brain samples (aged 34 to 51 years) were obtained from the National Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Page 9 of 11 Institute of Child Health and Human Development Brain and Tissue Bank for Developmental Disorders at the University of Maryland (Baltimore, MD) and were free from neurological disorders. Frozen brain samples from adult common chimpanzees, Pan troglodytes (aged 23 to 35 years), were obtained from the National Chim- panzee Brain Resource (Washington, DC). The chim- panzees had been cared for according to Federal and Institutional Animal Care and Use guidelines and died of natural causes. All brain tissue was collected with a postmortem interval of less than 8 h and stored at -80 ° C. Brain tissue was sampled from the ACC and CN from adult humans (n = 3) and chimpanzees (n = 3). Each sample was divided into two pieces: one for RNA- sequencing and one for quantitative proteomics. Librar- ies were constructed from poly-A-enriched RNA of 30 million 50-bp sequences. Orthologous gene models were constructed for each species, and sequences were mapped to species-specific genomes (hg19 and panTro3) [29, 30]. For the majority of the analyses, a dataset from human ACC was used which contained 815 transcript/ protein pairs. However, to assess the variation observed between species and brain regions the expression levels of 522 transcript/protein pairs from human and chim- panzee ACC and 499 from human and chimpanzee CN were analyzed. Both transcriptomic and proteomic data were normalized in edgeR [31]. To determine if the R2 values of the GO categories were better than a random sampling of transcripts and proteins, we performed permutation tests in which ran- dom transcripts and proteins were classified into cat- egories to mimic our observed data. The category sizes were sampled and replaced from the actual sizes of our observed categories for biological process, molecular function, and cellular component to ensure that the range of possible category sizes represented our dataset. The resampling occurred over 1000 iterations. Additional file 1: Complete list gene and protein expression in human ACC and the results of the OLS regressions. The file also contains the rank order changes of categories when R2 values are compared between species or regions of the brain. (XLSX 9357 kb) References 1. de Sousa Abreu R, Penalva LO, Marcotte EM, Vogel C. Global signatures of protein and mRNA expression levels. 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Abbreviations ACC: Anterior cingulate cortex; CN: Caudate nucleus; GO: Gene ontology; LC/ MS/MS: Liquid chromatography with tandem mass spectrometry; OLS: Ordinary least squares; PMI: Postmortem interval ACC: Anterior cingulate cortex; CN: Caudate nucleus; GO: Gene ontology; LC/ MS/MS: Liquid chromatography with tandem mass spectrometry; OLS: Ordinary least squares; PMI: Postmortem interval Page 10 of 11 Page 10 of 11 Page 10 of 11 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Bauernfeind and Babbitt BMC Genomics (2017) 18:322 Availability of data and materials 12. Wei Y-N, Hu H-Y, Xie G-C, Fu N, Ning Z-B, Zeng R, Khaitovich P. Transcript and protein expression decoupling reveals RNA binding proteins and miRNAs as potential modulators of human aging. Genome Biol. 2015;16:41. All data analyzed during this study are available through the supplemental files of Bauernfeind et al. [6] or upon request from the authors. 13. 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https://openalex.org/W1990459893	https://journals.iucr.org/e/issues/2011/12/00/vm2137/vm2137.pdf	English	null	5,5′-Bis(naphthalen-2-yl)-2,2′-bi(1,3,4-oxadiazole)	Acta crystallographica. Section E	2,011	cc-by	2,829	organic compounds Experimental Crystal data C24H14N4O2 Mr = 390.39 Monoclinic, P21=c a = 7.8982 (16) A˚ b = 5.7107 (11) A˚ c = 21.503 (5) A˚ = 109.82 (3) V = 912.4 (3) A˚ 3 Z = 2 Mo K radiation = 0.09 mm1 T = 293 K 0.18 0.14 0.12 mm Data collection Rigaku R-AXIS RAPID diffractometer Absorption correction: multi-scan (ABSCOR; Higashi, 1995) Tmin = 0.983, Tmax = 0.989 8518 measured reﬂections 2091 independent reﬂections 1468 reﬂections with I > 2(I) Rint = 0.030 Reﬁnement R[F 2 > 2(F 2)] = 0.039 wR(F 2) = 0.106 S = 1.07 2091 reﬂections 136 parameters H-atom parameters constrained max = 0.16 e A˚ 3 min = 0.18 e A˚ 3 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 Acta Crystallographica Section E Structure Reports Online ISSN 1600-5368 References Higashi, T. (1995). ABSCOR. Rigaku Corporation, Tokyo, Japan. Landis, C. A., Dhar, B., Lee, T., Sarjeant, A. & Katz, E. (2008). J. Phys. Chem. B, 112, 7939–7945. Haitao Wang,a Xiaoshi Jia,a Songnan Qu,b Binglian Baic and Min Lia* 8518 measured reﬂections 2091 independent reﬂections 1468 reﬂections with I > 2(I) Rint = 0.030 aKey Laboratory of Automobile Materials (MOE), College of Materials Science and Engineering, Jilin University, Changchun 130012, People’s Republic of China, bKey Laboratory of Excited State Processes, Changchun Institute of Optics, Fine Mechanics and Physics, Chinese Academy of Sciences, Changchun 130033, People’s Republic of China, and cCollege of Physics, Jilin University, Changchun 130012, People’s Republic of China Reﬁnement R[F 2 > 2(F 2)] = 0.039 wR(F 2) = 0.106 S = 1.07 2091 reﬂections 136 parameters H-atom parameters constrained max = 0.16 e A˚ 3 min = 0.18 e A˚ 3 Correspondence e-mail: minli@jlu.edu.cn Received 10 November 2011; accepted 15 November 2011 Received 10 November 2011; accepted 15 November 2011 Key indicators: single-crystal X-ray study; T = 293 K; mean (C–C) = 0.002 A˚; R factor = 0.039; wR factor = 0.106; data-to-parameter ratio = 15.4. Data collection: RAPID-AUTO (Rigaku, 1998); cell reﬁnement: RAPID-AUTO; data reduction: RAPID-AUTO; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to reﬁne structure: SHELXL97 (Sheldrick, 2008); molecular graphics: PLATON (Spek, 2009) and CrystalStructure (Rigaku/MSC, 2002); software used to prepare material for publication: SHELXL97. The title molecule, C24H14N4O2, lies on an inversion centre and the asymmetric unit containg one half-molecule. The naphthalene ring systems are twisted slightly with respect to the oxadiazole rings, making a dihedral angle of 1.36 (6). These molecules are -stacked along the crystallographic a axis, with an interplanar distance of 3.337 (1) A˚ . Adjacent molecules are slipped from the ‘ideal’ cofacial -stack in both the long and short molecular axis (the long molecular axis is deﬁned as the line through the naphthalene C atom in the 6- position and the molecular center, the short molecular axis is in the molecular plane perpendicular to it). The slip distance along the long molecular axis (S1) is 7.064 (1) A˚ , nearly a two- ring-length displacement. The side slip (S2, along the short molecular axis) is 1.159 (8) A˚ . We would like to thank Mrs Ye Ling and Dr Li Bao of Jilin University for the crystal structure analysis. This work was supported by the National Science Foundation of China (50873044, 51073071, 51103057, and 21072076) and Jilin University (200903014). Supplementary data and ﬁgures for this paper are available from the IUCr electronic archives (Reference: VM2137). Sci. 116, 143 164. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. Related literature Qu, S., Chen, X., Shao, X., Li, F., Zhang, H., Wang, H., Zhang, P., Yu, Z., W K., Wang, Y. & Li, M. (2008). J. Mater. Chem. 18, 3954–3964. For the synthesis of 1,3,4-oxadiazole derivatives: see Schulz et al. (1997). For related structures: see Schulz et al. (2005); Qu et al. (2008); Landis et al. (2008). g ( ) Rigaku (1998). RAPID-AUTO. Rigaku Corporation, Tokyo, Japan. ( ) Rigaku/MSC (2002). CrystalStructure. Rigaku/MSC Inc., The Woodlands, Texas, USA. Schulz, B., Bruma, M. & Brehmer, L. (1997). Adv. Mater. 9, 601–613. ( ) Schulz, B., Bruma, M. & Brehmer, L. (1997). Adv. Mater. 9, 601–613. Schulz, B., Orgzall, I., Freydank, A. & Xu, C. (2005). Adv. Colloid Interface Sci 116 143–164 , B., Bruma, M. & Brehmer, L. (1997). Adv. Mater. 9, 601–61 Schulz, B., Orgzall, I., Freydank, A. & Xu, C. (2005). Adv. Colloid Interface Sci. 116, 143–164. Sci. 116, 143–164. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. o3360 Wang et al. Acta Cryst. (2011). E67, o3360 doi:10.1107/S1600536811048513 supporting information S1. Comment Aromatic heterocycles, such as 1,3,4-oxadiazole and thiophene rings, which are conjugatable to phenyl rings, are often directly connected to the phenyl ring to obtain a large π-conjugated system or to tune the electronic structure. These compounds are of interest as charge transport materials or emitting layers in electroluminescent diodes (Schulz et al., 1997, Schulz et al., 2005). Comparing to thiophene derivatives, 1,3,4-oxadiazole derivatives are more likely to form π- stacked molecular packing (Schulz et al., 2005, Qu et al., 2008, Landis et al., 2008). 1, both 1,3,4-oxadiazole rings are in a trans-conformation, which yields a linear molecular shape. As shown in Fig. 1, both 1,3,4-oxadiazole rings are in a trans-conformation, which yields a linear molecular shape. These molecules are π-stacked along the crystallographic a-axis (Fig. 2). The molecules in the stacks are canted relative to the stacking axis by 26.57 (1)°. Adjacent molecules are slipped off each other in both long and short molecular axis to avoid unfavorable electrostatic interactions in the "ideal" cofacial stacks (Fig. 3). As shown in Fig. 1, both 1,3,4-oxadiazole rings are in a trans-conformation, which yields a linear molecular shape. These molecules are π-stacked along the crystallographic a-axis (Fig. 2). The molecules in the stacks are canted relative to the stacking axis by 26.57 (1)°. Adjacent molecules are slipped off each other in both long and short molecular axis to avoid unfavorable electrostatic interactions in the "ideal" cofacial stacks (Fig. 3). S2. Experimental supporting information Acta Cryst. (2011). E67, o3360 [https://doi.org/10.1107/S1600536811048513] 5,5′-Bis(naphthalen-2-yl)-2,2′-bi(1,3,4-oxadiazole) Haitao Wang, Xiaoshi Jia, Songnan Qu, Binglian Bai and Min Li Haitao Wang, Xiaoshi Jia, Songnan Qu, Binglian Bai and Min Li S1. Comment S2. Experimental The tile compound was synthesized through a two-step reaction. Firstly, naphthylacyl hydrazide was reacted with oxalyl chloride in THF at room temperature for 8 h, yielding the product, oxalyl acid N′,N′-di-naphthylacyl hydrazide. Secondly, the title compound was derived by intramolecular cyclization of this dihydrazide derivative with POCl3 under reflux conditions, and the coarse product was further purified by washing with DMSO for the 1H NMR FT—IR spectroscopic characterization and elemental analysis. Yield >70%. Crystals of the title compound suitable for X-ray diffraction were obtained by a slow diffusion method (diethyl ether was diffused into chloroform solution). S3 Refinement S3. Refinement Carbon-bound H-atoms were placed in calculated positions with C—H = 0.93 Å and were included in the refinement in the riding model with Uiso(H) = 1.2 Ueq(C). Figure 1 The molecular structure of the title compound with displacement ellipoids drawn at the 50% probability level. The asymmetric unit only contains a half molecule, the second half is generated by symmetry code -x, -y+1, -z+2. The line through C8 and the molecular center is defined as the long molecular axis. Carbon-bound H-atoms were placed in calculated positions with C—H = 0.93 Å and were included in the refinement in the riding model with Uiso(H) = 1.2 Ueq(C). Figure 1 g The molecular structure of the title compound with displacement ellipoids drawn at the 50% probability level. The asymmetric unit only contains a half molecule, the second half is generated by symmetry code -x, -y+1, -z+2. The line through C8 and the molecular center is defined as the long molecular axis. The molecular structure of the title compound with displacement ellipoids drawn at the 50% probability level. The asymmetric unit only contains a half molecule, the second half is generated by symmetry code -x, -y+1, -z+2. The line through C8 and the molecular center is defined as the long molecular axis. Acta Cryst. (2011). E67, o3360 sup-1 supporting information Figure 2 Molecular packing as viewed down the crystallographic a axis. Figure 3 Two adjacent molecules in the molecular stacks as viewed perpendicular to the molecular plane. The slip distances along the long molecular axis (S1) and short axis (S2) are 7.064 (1)Å and 1.159 (8) Å, respectively. Figure 2 Figure 2 Figure 2 g Molecular packing as viewed down the crystallographic a axis. g Molecular packing as viewed down the crystallographic a axis. Figure 3 supporting information Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.039 wR(F2) = 0.106 S = 1.07 2091 reflections 136 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0577P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 Δρmin = −0.18 e Å−3 Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.039 wR(F2) = 0.106 S = 1.07 2091 reflections 136 parameters 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0577P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 Δρmin = −0.18 e Å−3 Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites H-atom parameters constrained w = 1/[σ2(Fo2) + (0.0577P)2] where P = (Fo2 + 2Fc2)/3 (Δ/σ)max = 0.001 Δρmax = 0.16 e Å−3 Δρmin = −0.18 e Å−3 Figure 3 g Two adjacent molecules in the molecular stacks as viewed perpendicular to the molecular plane. The slip distances along the long molecular axis (S1) and short axis (S2) are 7.064 (1)Å and 1.159 (8) Å, respectively. g Two adjacent molecules in the molecular stacks as viewed perpendicular to the molecular plane. The slip distances along the long molecular axis (S1) and short axis (S2) are 7.064 (1)Å and 1.159 (8) Å, respectively. g Two adjacent molecules in the molecular stacks as viewed perpendicular to the molecular plane. The slip distances along the long molecular axis (S1) and short axis (S2) are 7.064 (1)Å and 1.159 (8) Å, respectively. 5,5′-Bis(naphthalen-2-yl)-2,2′-bi(1,3,4-oxadiazole) 5,5′-Bis(naphthalen-2-yl)-2,2′-bi(1,3,4-oxadiazole) Crystal data C24H14N4O2 Mr = 390.39 Monoclinic, P21/c Hall symbol: -P 2ybc a = 7.8982 (16) Å b = 5.7107 (11) Å c = 21.503 (5) Å β = 109.82 (3)° V = 912.4 (3) Å3 Z = 2 F(000) = 404 Dx = 1.421 Mg m−3 Mo Kα radiation, λ = 0.71073 Å µ = 0.09 mm−1 T = 293 K Block, colourless 0.18 × 0.14 × 0.12 mm Data collection Rigaku R-AXIS RAPID diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω scans Absorption correction: multi-scan (ABSCOR; Higashi, 1995) Tmin = 0.983, Tmax = 0.989 8518 measured reflections 2091 independent reflections 1468 reflections with I > 2σ(I) Rint = 0.030 θmax = 27.5°, θmin = 3.7° h = −10→10 k = −7→7 l = −27→27 Crystal data C24H14N4O2 Mr = 390.39 Monoclinic, P21/c Hall symbol: -P 2ybc a = 7.8982 (16) Å b = 5.7107 (11) Å c = 21.503 (5) Å β = 109.82 (3)° V = 912.4 (3) Å3 Z = 2 F(000) = 404 Dx = 1.421 Mg m−3 Mo Kα radiation, λ = 0.71073 Å µ = 0.09 mm−1 T = 293 K Block, colourless 0.18 × 0.14 × 0.12 mm sup-2 sup-2 Acta Cryst. (2011). E67, o3360 supporting information Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso/Ueq O1 0.18040 (11) 0.40921 (16) 0.97102 (4) 0.0412 (2) N1 0.01904 (14) 0.7350 (2) 0.94532 (6) 0.0482 (3) N2 0.14392 (14) 0.7347 (2) 0.91193 (6) 0.0479 (3) C1 0.04644 (15) 0.5425 (2) 0.97855 (6) 0.0407 (3) C2 0.23461 (16) 0.5413 (2) 0.92839 (6) 0.0388 (3) C3 0.38061 (15) 0.4564 (2) 0.90721 (6) 0.0373 (3) C4 0.46713 (17) 0.2407 (2) 0.93102 (6) 0.0448 (3) H4 0.4287 0.1499 0.9596 0.054 C5 0.60641 (17) 0.1660 (2) 0.91221 (7) 0.0453 (3) H5 0.6637 0.0256 0.9288 0.054* C6 0.66520 (15) 0.2993 (2) 0.86777 (6) 0.0389 (3) C7 0.80712 (17) 0.2254 (3) 0.84590 (7) 0.0506 (4) H7 0.8681 0.0868 0.8621 0.061* C8 0.85466 (18) 0.3556 (3) 0.80150 (8) 0.0586 (4) H8 0.9466 0.3037 0.7870 0.070* C9 0.76681 (19) 0.5674 (3) 0.77722 (8) 0.0565 (4) H9 0.8008 0.6543 0.7468 0.068* C10 0.63230 (17) 0.6463 (3) 0.79789 (6) 0.0459 (3) H10 0.5763 0.7881 0.7821 0.055* C11 0.57699 (15) 0.5144 (2) 0.84322 (6) 0.0364 (3) C12 0.43475 (15) 0.5888 (2) 0.86414 (6) 0.0379 (3) H12 0.3768 0.7297 0.8485 0.045* Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) x y z Uiso*/Ueq l atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) Acta Cryst. (2011). E67, o3360 sup-3 supporting information supporting information supporting information Atomic displacement parameters (Å2) U11 U22 U33 U12 U13 U23 O1 0.0390 (4) 0.0454 (6) 0.0423 (5) 0.0017 (4) 0.0180 (4) 0.0038 (4) N1 0.0447 (6) 0.0538 (7) 0.0506 (6) 0.0060 (5) 0.0222 (5) 0.0051 (6) N2 0.0464 (6) 0.0498 (7) 0.0518 (6) 0.0059 (5) 0.0225 (5) 0.0066 (5) C1 0.0343 (6) 0.0481 (8) 0.0402 (6) 0.0009 (5) 0.0133 (5) −0.0021 (6) C2 0.0392 (6) 0.0417 (7) 0.0358 (6) −0.0030 (5) 0.0131 (5) 0.0017 (5) C3 0.0363 (6) 0.0379 (7) 0.0371 (6) −0.0013 (5) 0.0115 (5) −0.0009 (5) C4 0.0512 (7) 0.0399 (7) 0.0453 (7) 0.0005 (6) 0.0190 (6) 0.0083 (6) C5 0.0481 (7) 0.0357 (7) 0.0495 (7) 0.0050 (5) 0.0130 (6) 0.0036 (6) C6 0.0366 (6) 0.0377 (7) 0.0403 (6) 0.0001 (5) 0.0103 (5) −0.0054 (5) C7 0.0433 (7) 0.0485 (9) 0.0588 (8) 0.0042 (6) 0.0156 (6) −0.0084 (7) C8 0.0438 (7) 0.0718 (11) 0.0680 (9) −0.0010 (7) 0.0290 (7) −0.0128 (8) C9 0.0522 (8) 0.0680 (11) 0.0569 (8) −0.0083 (7) 0.0285 (7) 0.0018 (8) C10 0.0450 (7) 0.0465 (8) 0.0472 (7) −0.0033 (6) 0.0168 (6) 0.0036 (6) C11 0.0363 (6) 0.0366 (7) 0.0355 (6) −0.0036 (5) 0.0110 (5) −0.0029 (5) C12 0.0395 (6) 0.0340 (7) 0.0391 (6) 0.0019 (5) 0.0118 (5) 0.0026 (5) Geometric parameters (Å, º) O1—C1 1.3568 (15) C6—C7 1.4190 (17) O1—C2 1.3636 (15) C6—C11 1.4228 (18) N1—C1 1.2889 (18) C7—C8 1.360 (2) N1—N2 1.4031 (16) C7—H7 0.9300 N2—C2 1.2986 (17) C8—C9 1.404 (2) C1—C1i 1.443 (3) C8—H8 0.9300 C2—C3 1.4588 (17) C9—C10 1.3600 (19) C3—C12 1.3714 (17) C9—H9 0.9300 C3—C4 1.4175 (18) C10—C11 1.4130 (18) C4—C5 1.3629 (19) C10—H10 0.9300 C4—H4 0.9300 C11—C12 1.4098 (17) C5—C6 1.4173 (19) C12—H12 0.9300 C5—H5 0.9300 C1—O1—C2 101.84 (10) C7—C6—C11 118.45 (12) C1—N1—N2 105.54 (11) C8—C7—C6 120.48 (14) C2—N2—N1 106.25 (11) C8—C7—H7 119.8 N1—C1—O1 113.78 (11) C6—C7—H7 119.8 N1—C1—C1i 127.93 (15) C7—C8—C9 120.87 (14) O1—C1—C1i 118.28 (15) C7—C8—H8 119.6 N2—C2—O1 112.58 (11) C9—C8—H8 119.6 N2—C2—C3 128.24 (12) C10—C9—C8 120.39 (14) O1—C2—C3 119.18 (12) C10—C9—H9 119.8 C12—C3—C4 120.03 (11) C8—C9—H9 119.8 C12—C3—C2 119.22 (12) C9—C10—C11 120.50 (14) C4—C3—C2 120.75 (12) C9—C10—H10 119.8 C5—C4—C3 120.24 (12) C11—C10—H10 119.8 Atomic displacement parameters (Å2) Acta Cryst. (2011). Symmetry code: (i) −x, −y+1, −z+2. supporting information E67, o3360 sup-4 supporting information C5—C4—H4 119.9 C12—C11—C10 121.71 (12) C3—C4—H4 119.9 C12—C11—C6 119.00 (11) C4—C5—C6 120.93 (13) C10—C11—C6 119.29 (11) C4—C5—H5 119.5 C3—C12—C11 120.93 (12) C6—C5—H5 119.5 C3—C12—H12 119.5 C5—C6—C7 122.68 (13) C11—C12—H12 119.5 C5—C6—C11 118.86 (11) C1—N1—N2—C2 0.01 (14) C4—C5—C6—C11 0.41 (19) N2—N1—C1—O1 0.15 (15) C5—C6—C7—C8 177.78 (13) N2—N1—C1—C1i 179.92 (16) C11—C6—C7—C8 −1.28 (19) C2—O1—C1—N1 −0.23 (14) C6—C7—C8—C9 1.1 (2) C2—O1—C1—C1i 179.97 (14) C7—C8—C9—C10 0.1 (2) N1—N2—C2—O1 −0.16 (14) C8—C9—C10—C11 −1.1 (2) N1—N2—C2—C3 −179.98 (12) C9—C10—C11—C12 −178.28 (12) C1—O1—C2—N2 0.24 (13) C9—C10—C11—C6 0.84 (19) C1—O1—C2—C3 −179.92 (11) C5—C6—C11—C12 0.36 (17) N2—C2—C3—C12 −0.4 (2) C7—C6—C11—C12 179.46 (11) O1—C2—C3—C12 179.76 (10) C5—C6—C11—C10 −178.78 (11) N2—C2—C3—C4 179.09 (12) C7—C6—C11—C10 0.33 (17) O1—C2—C3—C4 −0.72 (18) C4—C3—C12—C11 −0.42 (18) C12—C3—C4—C5 1.19 (19) C2—C3—C12—C11 179.11 (11) C2—C3—C4—C5 −178.33 (12) C10—C11—C12—C3 178.77 (11) C3—C4—C5—C6 −1.2 (2) C6—C11—C12—C3 −0.35 (18) C4—C5—C6—C7 −178.66 (12) sup-5 sup-5 Acta Cryst. (2011). E67, o3360
https://openalex.org/W3100251125	https://www.aclweb.org/anthology/2020.conll-1.40.pdf	English	null	Don’t Parse, Insert: Multilingual Semantic Parsing with Insertion Based Decoding	null	2,020	cc-by	7,700	Qile Zhu1∗, Haidar Khan2, Saleh Soltan2, Stephen Rawls2, and Wael Hamza2 1University of Florida, 2Amazon Alexa AI valder@ufl.edu {khhaida,ssoltan,sterawls,waelhamz}@amazon.com Qile Zhu1∗, Haidar Khan2, Saleh Soltan2, Stephen Rawls2, and Wael Hamza2 1University of Florida, 2Amazon Alexa AI valder@ufl.edu {khhaida,ssoltan,sterawls,waelhamz}@amazon.com Gupta et al. (2018) and Einolghozati et al. (2019) propose to use a Shift-Reduce parser based on Re- current Neural Network for these complex queries. Recently, Rongali et al. (2020) propose directly generating the parse as a formatted sequence and design a uniﬁed model based on sequence to se- quence generation and pointer networks. Their approach formulates the tagging problem into a generation task in which the target is constructed by combining all the necessary intents and slots in a ﬂat sequence with no restriction on the semantic parse schema. ∗Work done while interning at Amazon Alexa Abstract Semantic parsing is one of the key components of natural language understanding systems. A successful parse transforms an input utterance to an action that is easily understood by the system. Many algorithms have been proposed to solve this problem, from conventional rule- based or statistical slot-ﬁlling systems to shift- reduce based neural parsers. For complex pars- ing tasks, the state-of-the-art method is based on autoregressive sequence to sequence mod- els to generate the parse directly. This model is slow at inference time, generating parses in O(n) decoding steps (n is the length of the target sequence). In addition, we demonstrate that this method performs poorly in zero-shot cross-lingual transfer learning settings. In this paper, we propose a non-autoregressive parser which is based on the insertion transformer to overcome these two issues. Our approach 1) speeds up decoding by 3x while outperform- ing the autoregressive model and 2) signiﬁ- cantly improves cross-lingual transfer in the low-resource setting by 37% compared to au- toregressive baseline. We test our approach on three well-known monolingual datasets: ATIS, SNIPS and TOP. For cross lingual semantic parsing, we use the MultiATIS++ and the mul- tilingual TOP datasets. A relatively unexplored direction is the cross- lingual transfer problem (Duong et al., 2017; Su- santo and Lu, 2017), where the parsing system is trained in a high-resource language and transfered directly to a low-resource language (zero-shot). The state-of-the-art model leverages the autore- gressive decoder such as Transformer (Vaswani et al., 2017) and Long-Short-Term Memory (LSTM) (Hochreiter and Schmidhuber, 1997) to generate the target sequence (representing the parse) from left to right. The left to right autore- gressive generation constraint has two drawbacks: 1) generating a parse takes O(n) decoding time, where n is the length of the target sequence. This is further exacerbated when paired with standard search algorithms such as beam search. 2) In the cross-lingual setting, autoregressive parsers have difﬁculty transferring between languages. 2.1 Sequence Generation Via Insertion We begin by brieﬂy describing sequence generation via insertion, for a more complete description see (Stern et al., 2019). Let x1, x2, ..., xm be the source sequence with length m and y1, y2, ..., yn denotes the target se- quence with length n. We deﬁne the generated se- quence ht at decoding step t. In the autoregressive setting, ht = y1,2,...,t−1. In insertion based decod- ing, ht is a subsequence of the target sequence y that preserves order. For example, if the ﬁnal se- quence y = [A, B, C, D, E], then ht = [B, E] is a valid intermediate subsequence while ht = [C, A] is an invalid intermediate subsequence. During decoding step t + 1, we insert tokens into ht. In the previous example, there are three available insertion slots: before token B, between B and E and after E. We always add special tokens such as bos (begin of the sequence) and eos (end of the sequence) to the subsequences. The number of available insertion slots will be T −1 where T is the length of ht including bos and eos. If we insert one token in all available slots, multiple tokens can be generated in one time step. In summary, the main contributions of our work include: • To our knowledge, we are the ﬁrst to apply the non-autoregressive framework to the seman- tic parsing task. Experiments show that our approach can reduce the decoding steps by 66.7%. By starting generation with the whole source sequence, we can further reduce the number of decoding steps by 82.4%. In order to predict the token to insert in a slot, we form the representation for each insertion slot by pooling the representations of adjacent tokens. We have T −1 slots for a sequence with length T. Let r ∈RT×d, where T is the sequence length and d denotes the hidden size of the transformer decoder layer. All slots s ∈R(T−1)×d can be computed as: • We achieve new state-of-the-art Exact Match (EM) scores on ATIS (89.14), SNIPS (91.00) and TOP (86.74, single model) datasets. s = concat(r[1 :], r[: −1]) · Ws, (1) (1) • We introduce a copy encoder outputs mecha- nism and achieve a signiﬁcant improvement compared to the autoregressive decoder and sequence labeling on the zero-shot and few- shot setting in cross lingual transfer semantic parsing. 2.1 Sequence Generation Via Insertion Our approach surpasses the autore- gressive baseline by 9 EM points on average over both simple (MultiATIS++) and complex (multilingual TOP) queries and matches the performance of the sequence labeling baseline on MultiATIS++. where r[1 :] is the entire sequence representation excluding the ﬁrst token, r[: −1] is the entire se- quence representation excluding the last token and Ws ∈R2d×d is a trainable projection matrix. We apply softmax to the slot representations to obtain the token probabilities to insert at each slot. 2.2 Pretrained Models Pretrained language models (Devlin et al., 2019; Liu et al., 2019; Lan et al., 2020; Dong et al., 2019; Peters et al., 2018) have sparked signiﬁcant progress in a wide variety of natural language pro- cessing tasks. The basic idea of these models is to leverage the knowledge from large-scale cor- pora by using a language modeling objective to learn a representation for tokens and sentences. For downstream tasks, the learned representations are 1 Introduction Given a query, a semantic parsing module identiﬁes not only the intent (play music, book a ﬂight) of the query but also extracts necessary slots (entities) that further reﬁnes the action to perform (which song to play? Where or when to go?). A traditional rule- based or slot-ﬁlling system classiﬁes a query with one intent and tags each input token (Mesnil et al., 2013). However, supporting more complex queries that are composed of multiple intents and nested slots is a challenging problem (Gupta et al., 2018). A recent direction in machine translation and natural language generation to speed up sequence to sequence models is non-autoregressive decod- ing (Stern et al., 2019; Gu et al., 2018, 2019). Since the parsing task in the sequence to sequence frame- work only requires inserting tags rather than gener- ating the whole sequence, an insertion based parser is both faster and more natural for language transfer than an autoregressive parser. In this paper, we leverage insertion based se- 496 Proceedings of the 24th Conference on Computational Natural Language Learning, pages 496–506 Online, November 19-20, 2020. c⃝2020 Association for Computational Linguistics https://doi.org/10.18653/v1/P17 quence to sequence models for the semantic parsing problem that require only O(log(n)) decoding time to generate a parse. We enhance the insertion trans- former (Stern et al., 2019) with the pointer mech- anism, since the entities in the source sequence are ensured to appear in the target sequence. Our non-autoregressive based model can also boost the performance on the zero-shot and few-shot cross- lingual setting, in which the model is trained on a high-resource language and tested on low-resource languages. We also introduce a copy source mech- anism for the decoder to further improve the cross lingual transfer performance. In this way, the pointer embedding will be replaced by the corre- sponding outputs from the encoder. We test our proposed model on several well known datasets, TOP (Gupta et al., 2018), ATIS (Price, 1990), SNIPS (Coucke et al., 2018), MultiATIS++ (Xu et al., 2020) and multilingual TOP (Xia and Monti, 2021). 3.2.1 Pointer Network with Copy In this section, we introduce our non-autoregressive sequence to sequence model for the semantic pars- ing problem. Pointer Network: In the normal sequence to se- quence model, target tokens are generated by feed- ing the ﬁnal representations (decoder hidden states) through a feed-forward layer and applying a soft- max function over the whole target vocabulary. This is slow when the vocabulary size is large (Yang et al., 2018). In parsing, the entities in the source sequence will always appear in the target sequence. We can leverage the pointer mechanism (Vinyals et al., 2015) to reduce the target vocabulary size by dividing the vocabulary into two types: tokens that are the parsing symbols like intent and slot names, and pointers to words in the source sequence. 3.2 Insertion Transformer We use the insertion transformer (Stern et al., 2019) as the base framework for the decoder. The inser- tion transformer is a modiﬁcation of the original transformer decoder architecture (Vaswani et al., 2017). The original transformer decoder predicts the next token based on the previously generated sequence while the insertion transformer can pre- dict tokens for all the available slots. In this setup, tokens in the decoder side can attend to the entire sequence instead of only their left side. This means we remove the causal self-attention mask in the original decoder. 2 Background In this section, we introduce the sequence gener- ation via insertion operations and the pretrained models we leverage in our work. 497 Figure 1: Example of a simple query (left) and complex query (right). The complex query contains multiple intents and nested slots and can be represented as a tree structure. The two queries are represented as formatted sequences that are treated as the target sequence in the parsing task. IN is the intent, SL is the slot. Source tokens that appear in the target sequence are replaced by pointers with the form @n where n denotes its location in the source sequence. For complex queries, we can build the parse from top to bottom and left to right. drastically reduce the vocabulary size. We follow previous work and use symmetrical tags for intents and slots. Fig. 1 shows two examples, a simple query and a complex query with the corresponding target sequences. This formulation is also able to express other tagging problems like named entity recognition (NER). ﬁne-tuned for the task. This improvement is even more signiﬁcant when the downstream task has few labeled examples. We also follow this trend, and use the Trans- former (Vaswani et al., 2017) based pretrained lan- guage model. We use the RoBERTa base (Liu et al., 2019) (we refer to this model as RoBERTa) as our query encoder to fairly compare with the previous method. This model has the same archi- tecture as BERT base (Devlin et al., 2019) with several modiﬁcations during pretraining. It uses a dynamic masking scheme and removes the next sentence prediction task. RoBERTa is also trained with longer sentences and larger batch sizes with more training samples. For the multilingual zero- shot and few-shot semantic parsing task, we use XLM-R (Conneau et al., 2020) and multilingual BERT (Devlin et al., 2019) which are trained on text for more than 100 languages. 3.1 Query Formulation To train a sequence to sequence model, we prepare a source sequence and a target sequence. For the task of semantic parsing, the source sequence is the query in natural language. We construct the target sequence following Rongali et al. (2020) and Einolghozati et al. (2019). Tokens in the source sequence that are present in the target sequence are replaced with the special pointer token ptr-n, where n is the position of that token in the source sequence. By using pointers in the decoder, we can Since we have two kinds of target tokens, we use 498 two slightly different ways to obtain unnormalized probabilities for each type. For the tokens in the tagging vocabulary, we feed the hidden states gen- erated by the insertion transformer and slot pooling to a dense layer to produce the logits of size V (tag- ging vocabulary). The tagging vocabulary contains only the parse symbols like intents and slots to- gether with several special tokens such as bos, eos, the padding and unknown token. For the pointers, we compute the scaled dot product attention scores between the slot representation and the encoder output. The attention scores will be computed as The insertion transformer can do parallel de- coding since we can insert tokens in all available insertion slots. However, for each insertion slot, there may be multiple candidate tokens that can be inserted. For example, given a target sequence [A, B, C, D, E] and a valid subsequence [A, E], the candidates for the slot between token A and E are B, C, D. We use the two different weighting schemes proposed in Stern et al. (2019): uniform weights and balanced binary tree weights. Binary Tree Weights: The motivation for apply- ing binary tree weighting is to make the decoding time nearly O(log(n)). Consider the example of sequence A, B, C, D, E again, the desired order of generation would be [bos, eos] →[bos, C, eos] → [bos, A, C, E, eos] →[bos, A, B, C, D, E, eos]. To achieve this goal, we weight the candidates according to their positions. For the sequence above, candidates in the span of [bos, eos] are A, B, C, D, E. We assign token C the highest weight, then lower weights for B, D and the lowest weights for A, E. 3.1 Query Formulation a(Q, K) = QKT √ h , (2) (2) (2) where query (Q) is the slot representation, the encoder outputs would be the key (K) and h is the hidden size of the query. Since the hidden size of encoder and decoder may be different, we also do a projection of query and key to the same dimension with two dense layers. Notice that the length of attention scores follows the length of the source sequence. Concatenating the attention scores with size n and the logits for the tagging vocabulary (V), we get the unnormalized distribution over V + n tokens. We apply the softmax function to obtain the ﬁnal distribution over these tokens. Given a sampled subsequence with length k + 1, we have k insertion slots at location l = (0, 1, ..., k −1). Let cl0, ...cli be the candidates for one location l. We can deﬁne a distance function dj for each token j in the candidates of l: dl(j) = \|j −i 2\|, (3) (3) Copy Mechanism: Rongali et al. (2020) use a set of special embeddings to represent pointer tokens. This is a problem because the pointer embedding cannot encode semantic information since it points to different words across examples. Instead, we reuse the encoder output that the pointer token points to. Without copying, the special pointer embedding would learn a special position based representation for the source language that is hard to transfer to other languages. where i is the number of candidates in the location l. We then use the negative distance to compute the softmax based weighting (Rusu et al., 2016; Norouzi et al., 2016): wl(j) = exp(−dl(j)/τ) Pi m=0 exp(−dl(m)/τ) . (4) (4) Where τ is the temperature hyperparameter which allows us to control the sharpness of the weight distribution. Where τ is the temperature hyperparameter which allows us to control the sharpness of the weight distribution. 3.3 Training and Loss Uniform Weights: Instead of encouraging the model to follow a tree structure generation order, we can also treat the candidates equally. This per- forms better than the binary tree weights when we input the whole source sequence to the decoder as the initial sequence. In this case, we only need to insert the tagging tokens; the number of candidates is not as large as from scratch ([bos, eos]). This uniform weighting can be easily done by taking τ →∞. Training the insertion decoder requires sampling source and target sequences from the training data. We randomly sample valid subsequences from the target sequence to mimic intermediate insertion steps. We ﬁrst sample a length k ∈[0, n] for the subsequence, where n is the length of the target sequence (here n excludes the bos and eos tokens). We select k tokens from the target sequence and maintain the original ordering. This sampling helps the model learn to insert tokens from the initial generation state as well as intermediate generation. τ →∞. Loss Function:The autoregressive sequence to se- quence model uses the negative log-likelihood loss 499 3.4 Termination Strategy Terminating generation for insertion based decod- ing is not as straightforward as autoregressive de- coding, which only needs the no-insertion token to be predicted. Insertion decoding requires a similar mechanism for every insertion slot. When com- puting the slot-loss above, if there are no candi- dates for the slot we set the ground truth label as the no-insertion token. At inference time, we can stop decoding when all available slots predict the no-insertion token. However, there is a problem when combining the sampling method and this ter- mination strategy. The no-insertion token is more frequent compared with other tokens. The same situation is also encountered in (Stern et al., 2019). This is solved by adding a penalty hyperparame- ter to control the sequence length generated by the decoder. The hyperparameter is simply a scalar sub- tracted from the log probability of the no-insertion token for each insertion slot during inference. By doing this, we set a threshold for the difference between the no-insertion token and the second-best choice. 4.1 Datasets 4.1.1 SNIPS since in each decoding step, there is only one ground-truth label. However, in our approach, we have multiple candidates for each insertion slot. Therefore, we use the KL-divergence between the predicted token distribution and the ground truth distribution. Then the loss for insertion slot l is: The SNIPS dataset (Coucke et al., 2018) is a pub- lic dataset aimed to improve the semantic pars- ing models. It contains seven different intents: SearchCreativeWork, GetWeather, BookRestau- rant, PlayMusic, AddToPlaylist, RateBook, and SearchScreeningEvent. For each intent, there are about 2000 training samples and 100 test samples. The SNIPS dataset consists of only simple queries. Lslot(x, ht, l) = DKL((pl\|(x, ht))\|\|gl), (5) where pl is the distribution output by the decoder and gl is the target distribution where we set the probability to 0 for tokens that are not candidates. Note that the ground truth distribution depends on the weighting scheme for generation. 4.1.2 ATIS The Airline Travel Information System (ATIS) (Price, 1990) dataset was originally collected in the early 90s. The utterances are transcribed from the audio recordings of ﬂight reservation calls. Similar to SNIPS, it consists of only simple queries. ATIS contains seventeen different intents. However, nearly 70% of the queries are the FLIGHT intent. Finally, we have the complete loss averaged over all the insertion slots: L(x, ht) = 1 k + 1 k X l=0 Lslot(x, ht, l) (6) Recently, a multilingual version of ATIS called MultiATIS++ is introduced by Xu (2020). It is an extension of the Multilingual ATIS (Upadhyay et al., 2018). Besides the original three languages (English, Hindi and Turkish), MultiATIS++ adds six new languages including Spanish, German, Chi- nese, Japanese, Portuguese and French annotated by human experts and consists of a total of 37,084 training samples and 7,859 test samples. We ex- clude Turkish in our experiments as the test set size is limited. 4.1.3 TOP Since ATIS and SNIPS contain only simple queries, the Facebook Task Oriented Parsing (TOP) dataset (Gupta et al., 2018) was introduced for com- plex hierarchical and nested queries that are more challenging. The dataset contains around 45,000 annotated queries with 25 intents and 36 slots. They further split them into training (31,000), validation (5,000) and test (9,000). As shown in Fig. 1, the nested slots make it harder to parse using a simple sequence tagging model. We also do experiments on multilingual TOP (Xia and Monti, 2021) with Italian and Japanese data. In this dataset, the train- ing and validation set is machine translated while the test set is annotated by human experts. 4.2 Baseline Models In this section, we introduce the datasets and base- line models we experiment with. Then we report the results of monolingual experiments and cross lingual transfer learning experiments. Monolingual Baselines: For monolingual experi- ments, we select the algorithms reported in Ron- gali et al. (2020) as baselines for ATIS and SNIPS. 500 TOP ATIS SNIPS Method EM IC EM IC EM IC Joint BiRNN (Hakkani-T¨ur et al., 2016) - - 80.70 92.60 73.20 96.90 Attention BiRNN (Liu and Lane, 2016) - - 78.90 91.10 74.10 96.70 Slot Gated Full Attention (Goo et al., 2018) - - 82.20 93.60 75.50 97.00 CapsuleNlU (Zhang et al., 2019) - - 83.40 95.00 80.90 97.30 SR(S)+ELMO+SVMRank (Gupta et al., 2018) 83.93 - - - - - SR(E)+ELMO+SVMRank (Gupta et al., 2018) 87.25 - - - - - AR-S2S-PTR (paper) (Rongali et al., 2020) 86.67 98.13 87.12 97.42 87.14 98.00 AR-S2S-PTR (reproduce) (Rongali et al., 2020) 85.67 98.17 88.91 97.09 90.71 98.43 IT-S2S-PTR (τ = 1) 86.74 98.47 89.14 97.31 91.00 98.43 IT-S2S-PTR (input-src, uniform) 85.41 98.71 - - - - Table 1: Exact Match and Intent Classiﬁcation scores for on the test set. Input-src means the initial input of the decoder is the whole source sequence. For the shift reduce parsing models, E denotes the ensemble model and S is the single model. Table 1: Exact Match and Intent Classiﬁcation scores for on the test set. Input-src means the initial input of the decoder is the whole source sequence. For the shift reduce parsing models, E denotes the ensemble model and S is the single model. Table 1: Exact Match and Intent Classiﬁcation scores for on the test set. Input-src means the initial input of the decoder is the whole source sequence. For the shift reduce parsing models, E denotes the ensemble model and S is the single model. Model Avg. steps # tokens generated per step 1 2 3 4 5 6 7 8 9 AR-S2S-PTR 17.7 1 1 1 1 1 1 1 1 1 IT-S2S-PTR 5.9 1.0 2.0 3.96 6.66 6.24 3.17 1.6 1.4 1.2 IT-S2S-PTR(input-src) 3.1 4.99 2.92 1.37 1.00 0.54 0.27 0.25 1.0 1.0 Table 2: Decoding statistics on the TOP dataset. Average target sequence length of TOP is 17.7 tokens, we see that the insertion based parser can fully utilize binary tree decoding. ”input-src” means we set the whole source sequence as the initial decoder state. 4.2 Baseline Models Table 2: Decoding statistics on the TOP dataset. Average target sequence length of TOP is 17.7 tokens, we see that the insertion based parser can fully utilize binary tree decoding. ”input-src” means we set the whole source sequence as the initial decoder state. Table 2: Decoding statistics on the TOP dataset. Average target sequence length of TOP is 17.7 tokens, we see that the insertion based parser can fully utilize binary tree decoding. ”input-src” means we set the whole source sequence as the initial decoder state. match (EM) which requires all intents and slots are labeled correctly by the model. Two of them leverage the power of RNNs: with attention (Liu and Lane, 2016) and without atten- tion (Hakkani-T¨ur et al., 2016). Another model works completely with attention (Goo et al., 2018). A Capsule Networks based model is also in- cluded (Zhang et al., 2019). Finally, we compare with the autoregressive sequence to sequence with pointer model which is most recent (Rongali et al., 2020). Simple tagging based models cannot easily handle the complex queries in the TOP dataset. For the TOP dataset, we compare with two previous models, a shift reduce parsing model (Gupta et al., 2018) and the autoregressive sequence to sequence model (Rongali et al., 2020). For all monolingual experiments, we use RoBERTa as our pretrained encoder (Liu et al., 2019). 4.3.1 Model Conﬁguration We use the pretrained RoBERTa and mBERT as the encoder for our model. For the decoder side, we use 4 layers with 12 heads transformer decoder. The hidden size of the decoder is the same as the embedding size of the pretrained encoder. For op- timization, we use Adam (Kingma and Ba, 2015) with β1 = 0.9 and β2 = 0.98, paired with the Noam learning rate (initialized with 0.15) sched- uler (Vaswani et al., 2017) with 500 warmup steps. For cross-lingual experiments, we freeze the en- coder’s embedding layer. Cross lingual Baselines: For multilingual experi- ments (zero-shot and few-shot), we use a sequence labelling model based on multilingual BERT and an autoregressive sequence to sequence model (Ron- gali et al., 2020) as our baseline. To make fair comparasion, we also use the copy source mech- anism in the AR model. For sequence labeling, instead of using F1 score, we also use the exact 1Chinese is tokenized at the character level in mBERT, while Katakana/Hiragana are tokenized with whitespace. Data in MultiATIS++ is mixed in these two fashions. 4.3.2 Monolingual Results Table 4: Zero-shot EM scores on multilingual TOP dataset. Model is trained on English only. Table 4: Zero-shot EM scores on multilingual TOP dataset. Model is trained on English only. complex queries. It’s better to use the EM here for both simple and complex queries. We also report the intent classiﬁcation accuracy for our models. 4.3.2 Monolingual Results We use the exact match (EM) accuracy as the main metric to measure the performance of different models. By using EM, the entire parsing sequence predicted by the model has to match the reference sequence, since it’s not easy to apply the F1 score or semantic error rate (Thomson et al., 2012) to 501 en es pt de fr hi zh ja∗ avg IT-S2S-PTR 87.23 50.06 39.30 39.46 46.78 11.42 28.72 12.60 32.69 AR-S2S-PTR 86.83 40.72 33.38 34.00 17.22 7.45 23.74 10.04 23.77 mBERT 86.33 48.46 38.56 39.12 42.98 15.22 21.89 23.29 32.78 Table 3: Zero-shot cross lingual EM scores by our approach (IT), autoregressive baseline (AR) and sequence labeling baseline (mBERT). Results are averaged over four random seeds. For our approach, we initialize the decoder with source sequences. ∗indicates that the data format for the language is not consistent with the S2S model tokenizer. en es pt de fr hi zh ja∗ avg IT-S2S-PTR 87.23 50.06 39.30 39.46 46.78 11.42 28.72 12.60 32.69 AR-S2S-PTR 86.83 40.72 33.38 34.00 17.22 7.45 23.74 10.04 23.77 mBERT 86.33 48.46 38.56 39.12 42.98 15.22 21.89 23.29 32.78 Table 3: Zero-shot cross lingual EM scores by our approach (IT), autoregressive baseline (AR) and sequenc labeling baseline (mBERT) Results are averaged over four random seeds For our approach we initialize th Table 3: Zero-shot cross lingual EM scores by our approach (IT), autoregressive baseline (AR) and sequence labeling baseline (mBERT). Results are averaged over four random seeds. For our approach, we initialize the decoder with source sequences. ∗indicates that the data format for the language is not consistent with the S2S model tokenizer. en it ja IT-S2S-PTR 84.61 50.07 3.64 AR-S2S-PTR 85.4 41.06 0.64 Table 4: Zero-shot EM scores on multilingual TOP dataset. Model is trained on English only. that our approach can make full use of the paral- lel decoding during the ﬁrst three steps, since the average length of TOP’s test samples is only 17.7. Weighting Strategy: We do experiments on both binary tree weighting and uniform weighting for the TOP dataset. We set τ ∈[0.5, 1.0, 1.5, 2.0] and ﬁnd 1.0 performs best. Binary tree weights are better than uniform in the setting of decoding from scratch. However, uniform performs better when we decode from the whole source sequence. Table 4: Zero-shot EM scores on multilingual TOP dataset. Model is trained on English only. 4.3.3 Cross Lingual Transfer Results We also test on the multilingual TOP dataset (Xia and Monti, 2021), which extends the TOP datasets 502 IT-S2S-PTR AR-S2S-PTR # samples 0 10 50 100 0 10 50 100 it 50.07 50.13 52.69 56.42 41.06 42.23 44.98 46.96 ja 3.64 4.7 18.01 18.96 0.64 1.73 10.78 18.56 Table 5: Few-shot EM scores on multilingual TOP dataset with model pretrained on English. Training samples used in few-shot are sampled from the test set and excluded during testing. Table 5: Few-shot EM scores on multilingual TOP dataset with model pretrained on English. Training samples used in few-shot are sampled from the test set and excluded during testing. Table 5: Few-shot EM scores on multilingual TOP dataset with model pretrained on English. Training samples used in few-shot are sampled from the test set and excluded during testing. to other languages providing human annotated Ital- ian and Japanese test sets. TOP contains a much larger test set compared to ATIS. Table 4 shows the zero-shot results and Table 5 shows the few-shot results. EM IT-S2S-PTR 86.74 τ = 0.1 74.84 τ = 0.5 85.47 τ = 1.0 86.74 τ = 1.5 86.33 no copy 86.09 EM IT-S2S-PTR 86.74 τ = 0.1 74.84 τ = 0.5 85.47 τ = 1.0 86.74 τ = 1.5 86.33 no copy 86.09 In the zero-shot setting, our approach achieves 50.07 EM score for Italian while AR only achieves 41.06. Both models are unable to achieve good performance in the zero-shot setting for Japanese. We speculate on this behavior in the few-shot ex- periment results. Table 6: The ablation study for the τ parameter and copy source embedding vector vs. no copy in the mono- lingual setting. Results on the TOP dataset show the importance of copying source embeddings. We also ob- serve that small values of τ (i.e. weighting the central token for insertion heavily) degrade performance. In the few shot setting, we ﬁnetune the model in two stages, ﬁrst on the entire English data and then with 10, 50, 100 training samples from other languages. Our approach outperforms the AR base- line in all few shot settings. For Italian, increasing training samples from 10 to 100 does not result in much gain, since the knowledge from English can readily be transferred to Italian, probably due to the similarity of the languages. To further im- prove the performance on Italian, the model may need many more training samples. 4.3.3 Cross Lingual Transfer Results Main Result: Table 1 shows the results from monolingual experiments on three datasets: TOP, ATIS and SNIPS. Our insertion transformer with pointer achieves new state-of-the-art performance on ATIS and SNIPS under EM metric. For TOP dataset, our model matches the best performance reported for single models (AR-S2S-PTR) despite being 3x faster. For MultiATIS++, we train on English training data and test on all languages. Table 3 shows the results of our approach compared to the autoregressive and sequence labeling baselines. We ﬁnd that: • Our approach outperforms the baseline on most of the languages except Hindi and Japanese. For Japanese, we found inconsisten- cies in the tokenizer that is the likely cause of the degradation 1. We also experiment with starting generation with the entire source sequence as the initial state of the decoder. The performance degrades slightly in this case, possibly due to a training/inference mismatch in this setting. This degradation is likely due to training the model to generate the entire target se- quence but only asking the model to generate tags during inference. • The autoregressive baseline performs poorly on cross lingual experiments. For example, it only achieves 17.22 EM on the French test set while the other two systems achieve > 40 EM. This highlights the weakness of autore- gressive parsers that cannot produce parses directly from the encoded representations of the source sequence. Decoding Steps: Since our approach can do paral- lel decoding, the number of decoding steps is only O(log(n)). Table 2 shows the statistics for the av- erage decoding steps for the TOP dataset and the number of generated tokens per step. The inser- tion transformer with pointer only needs 5.9 steps while the autoregressive needs 17.7, resulting in a 3x speedup with insertion decoding. The decoding steps can be further reduced to 3.1 when we start decoding with the source sequence as the initial sequence for the decoder. Theoretically, a perfect binary tree based insertion model should generate 2n−1 tokens for the nth decoding step. We can see • The order of the sentence in Hindi and Japanese is different from others, this may limit the performance of transfer learning for S2S parsers. 4.3.3 Cross Lingual Transfer Results However, for Japanese little knowledge (like word order) can be transferred from English so both models can per- form as if training from scratch. There may be two reasons here: 1) the order of a sentence is different from English. 2) the annotated target is aligned with the original words in the multilingual TOP so the order of pointers are mixed. Thus, we see the EM scores improves drastically as the number of training samples increases. Models EM IT-S2S-PTR-Best 50.07 - copy 47.00 - input-src 42.03 AR-S2S-PTR-BEST 41.06 - copy 30.87 Table 7: The ablation study for source embedding copy- ing and starting generation from source tokens in the cross-lingual setting. Results are zero-shot in Italian. For the IT-S2S model, both copying and starting gener- ation with source tokens contribute to zero-shot perfor- mance Models EM IT-S2S-PTR-Best 50.07 - copy 47.00 - input-src 42.03 AR-S2S-PTR-BEST 41.06 - copy 30.87 Table 7: The ablation study for source embedding copy- ing and starting generation from source tokens in the cross-lingual setting. Results are zero-shot in Italian. For the IT-S2S model, both copying and starting gener- ation with source tokens contribute to zero-shot perfor- mance the best balance between equally weighting the can- didates and weighting the next token to be inserted heavily. However, we ﬁnd that when initializing the decoder with source sequences, uniform weights performs better than binary tree weights. Acknowledgements We thank the reviewers for their excellent feed- back. Special thanks also to Emilio Monti and Menglin Xia for their help with the multilingual TOP dataset. 5 Related Work Monolingual Semantic Parsing: The task ori- ented semantic parsing for intent classiﬁcation and slot detection is usually achieved by sequence la- beling. Normally, the system will ﬁrst classify the query based on the sentence level semantic and then label each word in the query. Conditional Random Fields (CRFs) (Peters et al., 2018; Lan et al., 2020; Jiao et al., 2006) is one of the most successful algorithms applied to this task before deep learning dominated the area. Deep learning algorithms boost the performance of semantic pars- ing, especially using recurrent neural networks (Liu and Lane, 2016; Hakkani-T¨ur et al., 2016). Other architectures are also explored, such as convolu- tional neural networks (Kim, 2014) and capsule networks (Zhang et al., 2019). 4.4 Ablation Study For ablation study, we separate the experiments to monolingual and multilingual as above. For multilingual experiments, we use the Italian from multilingual TOP dataset. For cross-lingual experiments, we introduce two components to improve the performance. Table 7 shows that both of them help in the zero-shot trans- fer setting. From the results, we can observe that initializing the decoder with the source sequence plays an important role in zero-shot transfer, which is impossible for the autoregressive based models. The copy mechanism is again beneﬁcial for both From Table 6, we observe that the copy mech- anism improves performance in the monolingual setting. For the hyperparameter τ, recall that a higher value for τ would result in ﬂatter (more uni- form) weights for the candidates. τ = 1.0 provides 503 the sequence to sequence models, improving the performance of even the autoregressive model from 30.87 EM to 41.06 EM in the zero-shot Italian ex- periment. puts, to mitigate these two problems. Our model can achieve O(log(n)) decoding time with parallel decoding. For the speciﬁc task of semantic pars- ing, we can further reduce the decoding steps by initializing the decoder sequence with the whole source sequence. Our model achieves new state-of- the-art performance on both simple queries (ATIS and SNIPS) and complex queries (TOP). In cross- lingual transfer, our approach surpasses the base- lines in the zero-shot setting by 9 EM points on average across 9 languages. References Alexis Conneau, Kartikay Khandelwal, Naman Goyal, Vishrav Chaudhary, Guillaume Wenzek, Francisco Guzm´an, Edouard Grave, Myle Ott, Luke Zettle- moyer, and Veselin Stoyanov. 2020. Unsupervised cross-lingual representation learning at scale. In Proceedings of the 58th Annual Meeting of the Asso- ciation for Computational Linguistics, pages 8440– 8451, Online. Association for Computational Lin- guistics. Cross Lingual Transfer Semantic Parsing: Mul- tilingual natural language understanding has been studied in a variety of tasks including part-of- speech (POS) tagging (Plank and Agi´c, 2018; Yarowsky et al., 2001; T¨ackstr¨om et al., 2013), named entity recognition (Zirikly and Hagiwara, 2015; Tsai et al., 2016; Xie et al., 2018) and se- mantic parsing (Xu et al., 2020). Before the ad- vent of pretrained cross-lingual language models, researchers leveraged the representations learned by multilingual neural machine translation (NMT). Another approach is to use NMT to translate be- tween the source language and the target language. However, it is challenging for the sequence tag- ging tasks: labels on the source language need to be projected on the translated sentences (Xu et al., 2020). Pretrained cross-lingual language models (Devlin et al., 2019; CONNEAU and Lam- ple, 2019) achieve great success in various multi- lingual natural language tasks. 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https://openalex.org/W4385487700	https://rbc.inca.gov.br/index.php/revista/article/download/3157/2013	Portuguese	null	O uso da Buprenorfina Sublingual (TEMGESIC) no Alívio da Dor em Pacientes com Câncer. Um Estudo em 32 Doentes	Revista Brasileira de Cancerologia	2,023	cc-by	3,238	Rev. Bras. Cancerol. 1988; 34 (2,3): 149-153 Rev. Bras. Cancerol. 1988; 34 (2,3): 149-153 149 Professor Titular do Departamento de Cirurgia da U.F.C. e membro do ICC - Agradecimentos: à Dra. Maria A. Cafferty e aos Laboratórios Reckitt & Colman; às Assistentes Sociais Raimunda Lindete Bessa dos Santos e Ana Célia Siébra de Queiroz. F.ndereço para correspondência: Av. Barão de Studart, 1001. Fortaleza, CE. CEP: 60120. Resumo Um estudo no controle da dor em doentes com câncer, através da Buprenorfina sublingual (Tem- gesic), foi realizado em 32 pacientes. Foram discutidos os problemas na avaliação c na exata mensuração da dor, tendo sido usadas três escalas para julgar a sua intensidade. Entre os paraefeitos da droga, os mais comuns foram: náuseas em 12,5%, vômitos em 21,8%, tonturas em 6,2% e sonolência em 15,6%. Dos 7 pacientes que apresentaram vômitos, 4 eram portadores de câncer gástrico ou de metástases hepáticas e tinham aquele sintoma antes de ini ciar o tratamento com o Temgesic. A sonolência foi mais freqüente em pacientes idosos, acima de 70 anos, os quais conseguiram diminuição daquele sintoma após reduzirem a dose de 20 mi- crogramas de 12/12 horas para 10 microgramas em intervalos de 6/6 horas. O efeito analgésico foi maior nos idosos; foi excelente nas metástases ósseas, muito bom tumores pélvicos e pouco satisfatório nos portadores de câncer gástrico. i i é i i i i n A aplicação sublingual, comparada com os analgésicos anteriormente aplicados por via paren- teral, mereceu maior aprovação dos doentes. Unitermos: buprenorfina: dor no câncer 0 uso da Buprenorfina Sublingual da Dor em Pacientes com Câncer. Doentes (TEMGESIC) no Alívio Um Estudo em 32 HAROLDO GODIM JUAÇABA Instituto do Câncer do Ceará (I.C.C.) - Fortaleza, CE Introdução modular a atividade dos caminhos ascendentes somá ticos. A própria introdução da injeção de opiáceos nos espaços epidural e subaracnóíde resultou do conheci mento da existência dos receptores daquele produto, localizados em maior quantidade na substância gela tinosa do corno meduiar dorsa\|24. Essa nova técnica tem mostrado ser efetiva^,7,18,25,30,34 e tem ganho ter reno sobre as operações neuroablativas e sobre as es timulações elétricas transcutâneas dos nervos3i. A dor tem sido uma companheira freqüente e mui tas vezes constante em 60 a 80% dos doentes de câncerS e cerca de 25% desses pacientes morrem sem ter conseguido alívio, mesmo pequeno, de suas doress. Os progressos na avaliação e no diagnóstico da dor crônica não acompanharam as teorias sobre o fenôme no nem tampouco as modalidades terapêuticas; a fi liação da dor a um evento fisiológico-sensorial exclu sivo ou ao resultado de um processo psicológico tem, segundo os psiquiatras, evitado valorizar outras relevan tes infoimações. Essa abordagem motivou a proposi ção de um Sistema de Avaliação Multiaxial da Dor que integraria os achados médicos, psicossociais e comportamentais32. Um novo termo — "Nocicepção" — foi criado pela Associação Internacional para o Es tudo da Dor "para significar" a percepção da dor e a sua graduação para o indivíduo23. A importância do fenômeno e a variedade de termos aplicados a situações de dor têm motivado a busca de correspondência de significação do seu grau de inten sidade entre algumas línguas^S; da mesma forma, o re conhecimento da dor e do seu alívio em recém-natos não é fácil e a similaridade básica do fenômeno, em ani mais, tem servido para uma melhor avaliação de sua intensidade e da aplicação correta da medicação20. A necessidade do conhecimento apropriado das dro gas analgésicas e narcóticas tem sido enfatizada e mes mo regulamentada por Associações Médicas, visando prover um melhor atendimento aos pacientes com dor crônicai5. A busca de novas drogas e de novos métodos para combater a dor tem sido beneficiada pelo melhor co nhecimento das rotas neuranatômiicas e da função de Rev. Bras. Cancerol, 34 (2,3): junho/setembro 1988 150 voso central através de mecanismo semelhante ao dos opiáceos endógenos23. A ação dos opiáceos exóge- nos no comportamento dos doentes tem tido interpre tação diferente quanto a sua origem: se da ação pró pria do remédio ou se secundária aos seus efeitos analgésicos27. Material e métodos A Buprenorfina é um derivado de tebaína. apresen tado sob a forma de um pó branco, levemente ácido e parcialmente solúvel em água. Ela tem uma potên cia analgésica 30 vezes maior que a morfina e é comer cializada sob a forma de comprimidos sublinguais com o nome de Temgesic^ e em injeções para uso intramus- cular ou venoso com o nome de Buprenex^. O Temge sic contém 200 microgramas, tem absorção rápida e seus efeitos são notados a partir de 10 minutos; uma série de publicações tern apresentado a sua ação e paraefeitos2,‘0,ii,i2.i4,27,29. o Buprenex contém 0,324mg/ml e é a única forma de Buprenorfina em uso atualmente nos Estados Unidos; ela está sendo anun ciada em Revistas Médicas como um. "analgésico de duração maior do que a da morfina e a da mieperidi- na"2. A buprenorfina é eliminada pelo fígado e o seu efei to é revertido parcialmente pelo naioxone (Narcan). Todos os 32 pacientes fcam atendidos inicialmen te no ambulatório, não tendo havido critério de esco lha. A queixa de dor era essencial para a admissão no estudo, assim como a insatisfação com os analgésicos que vinham tomando, uma vez que não era compara tivo o protocolo estabelecido. Foram excluídos os por tadores de insuficiência hepática, renal, respiratória e cardíaca graves, assim como os caquéticos, O apoio fa miliar para observar a administração correta do remé dio e para anotar os seus efeitos era essencial; houve também a participação ativa de assistente sociais. O efeito analgésico médio da buprenorfina é de 2,8 horas, estendendo-se a 7,2 horas. Os riscos de depen dência e os paraefeitos são menores dos que os da morfina^. O exato mecanismo da sua ação analgésica parece ser resultante da grande afinidade de conjuga ção com os opiáceos do sistema nervoso centra!, a en- dorfina e a dinorfina, dois dos mais potentes inibido res da nocicepção; a buprenorfina, como opiáceo exó- geno, em dose apropriada, produziría a supressão da atividade transmissora nos neurônios do sistema ner- Um questionário sobre a medicação analgésica que vinha sendo tomada anteriormente e os seus efeitos era preenchido, tentando-se avaliar também o limiar da dor de cada paciente. O período de 2 semanas ficou estabelecido como prazo mínimo necessário para a avaliação dos resulta dos e dos paraefeitos do Temgesic. Material e métodos Não houve conco mitância de outro tratamento, cirúrgico, químico ou de irradiação: todos os pacientes já tinham feito um ou Figura 1 — Parte da ficha de anotações 8 9 10 6 7 2 3 5 0 1 4 Escala I Antes do Temgesic Nenhuma dor Dor insuportável Efeito excelente Nenhum efeito 0 = 10 = 10 = Após o Temgesic 0 = Escala II — (Dor antes do Temgesic) 02 Q 06 05 04 03 01 1. Ausência da dor 2. Dor presente mas facilmente ignorada 3. Dor que não pode ser ignorada mas não interfere com as atividades normais 4. Dor que interfere com a concentração 5. Dor que interfere com as atividades normais, mas o doente exerce os cuidados básicos (alimentação e higiene) 6. Dor que interfere com todas as atividades Escala III - (Dor antes do Temgesic) 04 02 03 01 1. Sem dot 2. Alguma dor 3. Dor considerável 4. Dor insuportável TEMGESIC na dor do câncer: Juaçaba 151 mais daqueles tratamentos e tinham doença avança da. gas Analgésicas no Tratamento da Dor no Câncer" de que a Buprenorfina poderia provocar depressão respi ratória e que o naioxone tinha somente uma ação re- versiva parcial sobre a drogai^; entretanto, um estudo prospectivo sobre a Buprenorfina sublingual (Temge sic) em pacientes idosos não referiu aquela complica ção e os efeitos colaterais mais comuns foram: náuseas (15,7%), vômitos (9,8%) e tonturas (5,9%)22. Em ou tro trabalho empregando a forma parenteral (Buprenex) não houve nenhum caso de depressão respiratóriass, como também em todos os trabalhos citados anterior mente. Q naioxone tem demonstrado uma ação para doxal: quando aplicado conjuntamente com narcóticos, em dose apropriada, não diminui a sua ação analgési ca e tem tornado menor o risco de dependência. Esse Poram adotadas 3 escalas de medição da intensidade da dor, do trabalho de Jensenie. A Figura 1 mostra aquelas escalas e os seus parâmetros. Foi instituído o regime de 1 comprimido de Temge- sic com 20 microgramas, de 12/12 horas, nos dois ou três primeiros dias, aumentando-se para 1 comprimi do de 8/8 horas se a analgesia fosse insuficiente; em alguns casos em que a sonolência se manifestava, a do se de 1/2 comprimido de 6/6 ou de 4/4 horas pro vou ser efetiva especialmente em pacientes idosos. Material e métodos A medicação era suprida para 7 dias e uma nova ava liação feita; os paraefeitos anotados e o regime, modi ficado ou mantido, de acordo com o resultado da res posta à dor. Nenhum paciente desistiu da medicação por algum paraefeito ou por sua completa ineficácia; alguns tive ram sua doença em progresso rápido e não completa ram os 14 dias necessários para avaliação e por essa razão não foram incluídos no estudo. Quadro I - Distribuição dos casos segundo faixas etárias Quadro I - Distribuição dos casos segundo faixas etárias Idade (em anos) N? de casos 20-29 2 30-39 2 40^9 50-59 60-69 70-79 8 1 12 5 80-1- 2 Total 32 O sexo, a faixa etária e a relação das patologias com os respectivos paraefeitos estão listados nos Quadros I e II. A graduação da dor nas diversas patologias e o resultado analgésico com o Temgesic são apresenta dos no Quadro III. Uniterms: buprenorphine: câncer pain Resultados e comentários Uma das preocupações com o trabalho foi a asser tiva em artigo sobre os "Princípios e Práticas das Dro- Quadro II — Patologia e paraefeitos Quadro II — Patologia e paraefeitos Paraefeitos Total de casos Diagnóstico/localização Náuseas sem vômitos Vômitos Tonturas Sonolência Mama com metástase óssea Mama com metástase hepática Mama com recidiva no plastrão Colo uterino Endométrio Ovário Estômago Cólon com metástase hepática e peritoneal Esôfago Próstata Pulmão Pele-Carcinoma espinocelular da face Melanoma cutâneo maligno Nasofaringe Fibrossarcoma Neurofibrossarcoma 7 T 2 1 1 3 1 1 1 1 1 4 2 3 1 2 2 1 5 1 1 1 1 1 1 1 1 1 1 Total 32 4(12,5%) 7(21,8%) 2(6,2%) 5(15,6%) Diagnóstico/localização Rev. Bras. Cancerol. 34 12,3): junho/setembro 1988 152 Quadro III — Intensidade da dor e resposta ao Temgesic dos 32 casos observados segundo as escalas.1 e II Escala I — (Efeitos) Escala II — (Dor) Excelente Bom Regular Mau Graduação Diagnóstico / Localização 8 9 10 5 6 1 2 3 4 5 6 7 1 2 3 4 2 1 2 5 Mama com metástase óssea — — Mama com metástase hepática — — Mama com recidiva no plastrão — — Colo uterino — — Endométrio — — Ovário — — Estômago — — Cólon com metástase hepática e peritoneal — — Esôfago — — Próstata — — Pulmão — — Pele-Carcinoma espinoceluiar da face — — Melanoma cutâneo maligno — — Nasofaringe — — Fibrossarcoma — — Neurofibrossarcoma — — 1 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 2 2 2 2 1 1 2 1 1 1 1 2 2 3 1 1 1 1 1 1 1 1 1 1 1 1 1 7 14 3 9,3% 3,1% 12,5% 21,8% 44% 9,3% 3 1 4 8 7 17 Total de casos 25% 21,2% 53,1% — Intensidade da dor e resposta ao Temgesic dos 32 casos observados segundo as escalas.1 e II parativo com a Pentazocina (Taiwin); esse produto re velou ser menos atuante quanto ao sono normal do que a Buprenorfinaii. fantasma da dependência tem sido um dos maiores res ponsáveis pelo "subtratamento" da doris e esse receio de transformar em ad ictos, doentes em tratamento de dor no câncer, tem sido contestado26. Summary Os estudos sobre processos terapêuticos são sem pre vulneráveis, caso sejam consideradas como parâ metro a sua eficácia e “as duas principais ameaças à sua validade são a incerteza e a parciaiidade"2i. Quan do as observações colhidas se apoiam em dados sub jetivos, como no presente caso, o ajustamento das in formações às variáveis de importância não é fáciM^. /n order to evaluate control of câncer pain undersub- lingual Buprenorphine (Temgesic), 32 patients were evaluated. The difficulties in measurement of clinicai pain in- tensity are discussed. Three scales were used in the criteria for judging pain intensity. Neste estudo a tolerância ao Temgesic foi boa; ne nhum doente recusou tomar a medicação e nos ido sos o seu efeito pareceu melhor: a fragmentação da do se com menor espaço de sua aplicação não prejudicou a analgesia. Essa melhor ação nos pacientes idosos tem sido observada com outros narcóticosN^. Entre os pa- raeíeitos, as náuseas e vômitos foram mais freqüentes nos doentes de câncer gástrico e nos portadores de me- tástases hepáticas ou peritoneais: dos 7 doentes que apresentaram vômitos somente 2 não eram portado res daqueles tumores e 80% deles tinham vômitos an tes de iniciarem o Temgesic. The most common side effects were: nausea in 12.5%, vomiting in 21.8%, dizziness in 6.2%, and drowsiness in 15.6%. Of the 7 patients with vomits only 3 did not have the symptom before starting on Temges ic. Therewasnocaseofrespiratorydepression. Drowsi ness, more common in patients over 70 years, benefict- ed from smaíler doses (10 micrograms) at 6 hour inter- vals instead of full doses of 20 micrograms at 12 hour intervals. The analgesic effect was enhanced by aging; it was excellent in metastatic bone disease, very good in pel- vic tumorsand less than satisfàctory in gastric câncer. Os resultados sobre a dor foram muito bons, princi palmente nos portadores de lesões ósseas metastáti- cas e de tumores pélvicos. A sonolência que foi referi da por 5 pacientes, em interrogatório mais detalhado, mostrou ter sido confundida em alguns casos com "mais horas de sono" fato já avaliado em trabalho com- y g The sublingual route was appreciated byall patients, compared with previous parenteral use of analgesic drugs. TEMGESIC na dor do câncer: Juaçaba 153 Referências Bibliográficas 17. Kaiko RF, Wallenstein SL, Bogers AG, Grabinski PY, Houde RW - Narcotic in the elderly. Med Clin North Am, 1982; 66: 1079-89. 18. Summary Leavens ME, HilI CS, Cech DA, Weyland JB, Weston JS - Intrathecal and in- traventricular morphine for pain in câncer patients: initial study. J Neurosurg, 1982: 56: 241-5. 1. Bellville JW, Forrest JrWA, MillerE, Brown JrB — Influence of age on pain relief from analgesics. JAMA, 1971; 217: 183541. 19. MarksRM,SacherEJ - Undertreatmentof medicai inpalientswith narcotic anal gésica. Ann Intern Med, 1973: 78: 173-81. 2. Bond MR - Dor-Natureza, análisee tratamento. 2ed. Rio de Janeiro, Colina, 1986' p. 181. 20. Morton DB - Assessment of pain. Vet Rec, 1986: 119:435. 20. Morton DB - Assessment of pain. Vet Rec, 1986: 119:435. 3. Buprenex. Linger Acting Analgesia than Morphine or Meperidine (advertise). Arch Surg, 1986: 121:225-7. 21. MosesLE - The series of consecutiva cases as a device for ass 21. MosesLE - The series of consecutiva cases as a device for assessing outcomes of intervention. (Statistic in practice.) N EngI J Med, 1984: 311 (11): 705-10. 4. Buprenex. In: Physicians Desk Reference Book. New Jersey. Medicai Econom- ics Co Inc Cradell, 1987; p. 1408-10. 22. Nassar MA, McLeavy MA, Knox J — An open study ofsublingual buprenorphine in the treatmentofchronic pain inthe elderly. Curr Med ResOpin, 1986:10:251-5. 5. Buprenorphine ITemgesic) In: British National Fbrmulary. London British Medi cai Association and Pharmaceutical Society of Great Britain, 1984; p. 165. 23. Payne R - Anatomy, physiology and neuropharmacology of câncer pain. Med Clin North Am, 1987; 71: 153-67. i Association and Pharmaceutical Society of Great Britain, 1984; p 6. Coombs DW, Saunders RL, Gaylor MJ — Relief of continuous chronic pain by intraspinal narcotic infusion via an implantable reservoir. JAMA, 1983; 250:223-6. 24. Payne R - Role of epidural and intrathecal narcotics and peptides in the manage- ment of câncer pain. Med Clin North Am, 1987; 71: 313-27. 7. Cousins MJ, Mather LE — Intrathecal and epidural administrations of opioids. Anesthesiology, 1984; 61: 276-3'l0. 25. Penn RD, Paige TA, Gottschalk W, Ivanizovich AD - Câncer pain relief using chronic morphine infusion. J Neurosurg, 1984; 61: 302-6. chronic morphine infusion. J Neurosurg, 1984; 61: 302-6. 8. Coyle N - A model of continuity of care for câncer patients with chronic pain. Med Clin North Am, 1987; 71: 259-90. 26. Porter J, Jyck H - Addiction rare in patients with narcotics. N EngI J Med, 1980; 302: 123. 9. Daudt RC, Cleeland CS — Theprevalenceand severity of pain in câncer Câncer 1982: 50: 1913-19. 27. Summary Price DD, Gruen AV, Miller J - Psychophysical analysis of morphine analgesia. Pain, 1985: 22: 261-9. 10. DeConno F Ripamonti C, Tamburini M, Ventafndda V - Buprenorfina nel dolo- re da cancro: confronto incrociato con Ia pentazocina. Minerva Med, 1987; 78' 1177-81. 28. Radvila A, Adier RH, Galeazzi RL, Vorkauf H - The development of a german language (Bernel pain questionaire and its appiication in a situation causing pain. Pain, 1987: 28: 185-95. 11. Dini D, FassioT, Gottlieb A, Gini M — Studio controllato sulLefetto analgésico e sulla tolerabilità delia buprenorfina, in pazienti da neopiasia. Minerva Med, 1986; 77: 93-104. 29. Robie DS — A trial of sublingual buprenorphine in câncer pain. Br J Clin Phar- macol, 1979: 7: 3158-75. 30. 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Vanacker B, Vandermeersch E, Tomassen J - Comparison of intramuscular buprenorphine and a buprenorphine naioxane combination in the treatment of postoperative pain. Curr Med Res Opin, 1986; 10: 139-44. 15. Health and Public Policy Committee. American College of Physicians. Drug ther apy (br severe chronic pain in terminal illness. Ann Intern Med, 1983; 99:870-3. 15. Health and Public Policy Committee. American College of Physic apy (br severe chronic pain in terminal illness. Ann Intern Med, 19 postoperative pain. Curr Med Res Opin, 1986; 10: 139-44. 16. Jensen MP, Karoly P, Braver S — The measurement of clinicai pain intensity: a comparison of six methods. Pain, 1986; 27: 117-26. 34. Zenz M, Schappier-Scheele B, Neuhaus R, Piepenbrock S, Hilfrich D - Long term epidural morphine analgesia in câncer pain. Lancet, 1981; 1:91. Corrigenda No trabalho "Forma Rabdomiornatosa do Tumor de Wilms. Relato de um Caso com Envolvi mento Bilateral", de Bittencourt ecols., da Revista Brasileira de Cancerologia, Vol. 34 n.1 às pági nas 16, onde se lê "Analisando 15 casos de longa evolução" leia-se "Analisando 15 casos da lite ratura e cinco seus, Wigger verificou que esta variante apresenta-se sob forma de tumores vo lumosos de longa evolução"... A Editoria
https://openalex.org/W3139200523	https://www.e3s-conferences.org/10.1051/e3sconf/202124405015/pdf	English	null	Formation and development of technotopia as a concept of the figurative and stylistic direction of innovative architectural activity	E3S web of conferences	2,021	cc-by	2,723	* Corresponding author: perspektiva-aa@mail.ru Formation and development of technotopia as a concept of the figurative and stylistic direction of innovative architectural activity Alexander Pleshivtsev1,* and Tatiana Pakunova1 1 State University of land management, 105064, 15, Kazakova str., Moscow, Russia 1 State University of land management, 105064, 15, Kazakova str., Moscow, Russia Abstract. Purpose: to analyze the modern display of utopian ideas in architectural activity for the development of innovative directions of organization and forming of the object environment, to identify and define the features of technotopia as a figurative and stylistic direction that combines idealistic and pragmatic principles in architectural creativity. Methods and materials: empirical and theoretical methods of analogy and abstraction focused on analyzing the properties, possibilities and conditions that determine the features of utopian ideas displayed in architectural creativity. A method of generalizing the experience of theoretical knowledge and development in the implementation of innovative technological solutions for architectural objects. Results: A two-level structure of signs of utopia in architectural creativity was developed. An algorithm for transforming ideas about an ideal world is proposed, taking into account the possibilities of practical implementation of technological priorities in architectural activity. The properties of technotopia as a figurative and stylistic direction characterizing the features of the practical implementation of technological priorities of architectural activity have been confirmed. Conclusions: The study of the nature of utopias is one of the key topics and opportunities for the development of promising figurative and stylistic directions in architectural creativity. The fundamental idealistic (utopian) principle of architectural creativity is capable of evolution through integration with the results and possibilities of technological progress of various fields of knowledge. Technological priorities in the organization of architectural space make it possible to significantly expand the possibilities of traditional compositional techniques in solving applied and practical problems of architectural activity. © 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/). E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124405015 1 Idealistic and pragmatic principles in modern architectural activity Each architectural concept (image, project) contains an idealistic (utopian) and pragmatic principle in an attempt to organize and display the subject-spatial environment. The utopian beginning of architectural activity reflects a certain “super idea”, “ideal © 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/). E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124405015 image”, “metaphysics of architecture”. The term “utopia” translated from Greek (οὐ “not” and τόπος “place” or εύ “good”) means: “... a blessed country or a non-existent place” and denotes a non-existent ideal, which, nevertheless, can be approached [1,2,3]. image”, “metaphysics of architecture”. The term “utopia” translated from Greek (οὐ “not” and τόπος “place” or εύ “good”) means: “... a blessed country or a non-existent place” and denotes a non-existent ideal, which, nevertheless, can be approached [1,2,3]. Nowadays, due to the lack of a precise definition of a single set of features and criteria, the term “utopia” is blurred and is used in relation to almost all large-scale projects (activities). Utopia, as one of the possible types of creative activity, is characterized by a set of stable features and display features. In a commonly used sense, the concept (term) “utopia” has become widespread for the semantic identification of conditions and/or a lack of prerequisites for the implementation of large-scale and difficult-to-implement projects or forecasts in various areas of creative or practical activity. Moreover, it is the “scale” characteristic that is taken as a negatively accentuated property of projects in order to be called utopian projects or events identified with creative processes and transformations of material and non-material space. This approach comes into conflict with the creative and fundamentally realizable logic and practical results of architectural creativity. Architecture, being a creative, positive and programmatic type of theoretical and pragmatic activity, is inextricably linked with the development of utopian thought and creativity, starting from the historical eras of Antiquity and the Renaissance [1,4,5,6]. The genesis of the development of utopian ideas includes successive stages of development: from the search for a romantic image to the formation of their purely pragmatic ideas. The balance saturation and proportional distribution of the utopian and pragmatic principles are different for each architectural image, but they are necessarily present in each project (subject of architectural creativity). 1 Idealistic and pragmatic principles in modern architectural activity The pragmatic beginning is the “physical” compliance with technological, functional, structural, operational criteria and conditions. Progress in the development of resources and opportunities for creative activities, primarily intellectual, technical and technological ones, prompts intentions in the development and implementation of the architectural ideal here and now. For this reason, the creative genre of utopia has transformed from abstract, theoretical reasoning into the subject of searches for applied recipes and solutions in architectural activity. The utopian idea in architecture is a representation of the ideal organization of the surrounding subject-spatial environment, and the utopia contains the figurative, fundamental basis of architectural creativity. Architectural utopia can be considered as a way of reflecting the principles of idealized consciousness in the formation of an architectural work, implying the presence of a “creative composition of an ideal image”, which is proposed as a subjective form of representation of the desired reality [7,8,9]. The combination of methods of abstract philosophy and practical creation reflects the main essence of architectural activity, and, in fact, architecture is one of the most significant tools for finding and organizing ideal space and harmony. Understanding the peculiarities of the processes of formation and transformation of utopian ideas, the cyclical and paradoxical nature of these processes, understanding the reasons for their appearance and their possible consequences will allow a deeper understanding of the essence of the creative process, the reasons and patterns of manifestation of architectural thought, as well as predict the main development trends and results of displaying architectural creativity [7, 10]. To assess architectural projects and determine common structural features an relationships, a two-level structure of utopia features can be adopted: − main signs: o novelty; o radicalism; o totality (due to its logical nature); 2 E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124405015 o timelessness (lack of spatial development, changes in time); o autarky (self-sufficiency and self-dependence, spatial isolation); y ( y p p o determinism or limitation of the development process; o hierarchical subordination and regulations for social processes and phenomena. − indirect (secondary) signs: o hierarchical subordination and regulations for social processes and phenomena. 1 Idealistic and pragmatic principles in modern architectural activity − indirect (secondary) signs: − indirect (secondary) signs: o presence of a dominant style; o the use of an excessive, hyperbolized scale (“gigantism”) as the main means of displaying the features of the work; o the use of an excessive, hyperbolized scale (“gigantism”) as the main means of displaying the features of the work; o monotony and like-mindedness; o lack of internal conflicts. Topical issues and modern trends, containing the characteristic features of architectural utopias, are much more complex phenomena than their representations for past historical eras. The social and scientific role of an architectural utopia is controversial and is largely determined by the conjuncture of a specific situation. The peculiarity of the current state and trends of architectural utopia lies, first of all, in the presence of the necessary conditions for conducting analytical studies of the genesis of the forms and content of the type of consciousness under consideration, in accordance with the characteristic features of the corresponding era, and allows for the possibility of making predictive assessments of promising directions for the development of style trends. 2 The concept and possibilities of technological formation of the subject-spatial environment of the architectural space From the total mass of modern utopian projects, one of the most noticeable directions stands out, which is evolutionarily formed through the integration of utopian representations of the ideal world of previous historical eras and the technological capabilities of the present time. The technotopia direction is focused on the implementation of technological priorities (including those that are not currently available) in existing architectural projects for new and existing objects [11,12]. One of the fundamental points in the formation of the methodological base of technotopia is the fact of continuity: it not only does not deny, but rather complements the theoretical concepts of architectural utopia, including their most important achievements, expands the subject of analysis, allows a new look at the processes of evolution and transformation of architectural space and corresponding architectural images. The signs of modern technotopia in a categorical (and more often grotesque, intolerant to the experience of the history of the development of architectural science) form reflect the features and possibilities of implementing unconventional compositional solutions supported by the appropriate technological performance [13, 14, 15]. Figure 1 shows examples of compositional and technological solutions that reflect the modern representation of the interaction of the form and the functional content of architectural images through the methods and means of technotopia. 3 3 E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124405015 a) Statoil International Offices, Norway [16] b) ING House, Nederland [17] c) Kubuswoningen, Nederland [18] d) Vodafone Headquarters, Portugal [19] Fig. 1. Features of shaping expressed by means and techniques of technotopia. a) Statoil International Offices, Norway [16] a) Statoil International Offices, Norway [16] b) b) ING House, Nederland [17] d) Vodafone Headquarters, Portugal [19] a) S a o e a o a O ces, No way [ 6] c) Kubuswoningen, Nederland [18] d) Vodafone Headquarters, Portugal [19] c) Kubuswoningen, Nederland [18] Fig. 1. Features of shaping expressed by means and techniques of technotopia. Fig. 1. Features of shaping expressed by means and techniques of technotopia. For the considered architectural images, a creative (in a certain sense, utopian) idea allows finding a correct, rational and aesthetic solution that will reflect the set of functional, technological and design requirements necessary to harmonize interaction with the environment. The field of architectural creativity will remain a field of search for an ideal idea of the surrounding space and its main tool for its formation. 1. A.V. Ikonnikov, Architecture of the 20th century. Utopias and reality. Volume II (Progress-Tradition, M., 2002) 5 Conclusion Architectural activity for the formation, maintenance and transformation of the content of the surrounding subject space is presented as a complex synthesis of artistic (utopian principle) and technological (pragmatic principle) processes, the result of which reflects a special, synthesized and integrated approach to solving situational and promising tasks aimed at ensuring conditions of functional efficiency, safety and comfort of the architectural environment. The technological component (production effectiveness) of the formation of an architectural image is becoming an increasingly significant structural element of modern architectural and construction culture, evolution and harmonization of architectural knowledge. 2 The concept and possibilities of technological formation of the subject-spatial environment of the architectural space The formation of modern samples of the figurative and stylistic direction of technotopia should be considered as alternative methods of geometric and tectonic proportionality of traditional forms, accompanied by the evolution of known parameters and revolutionary organization of new properties of materials, tectonic features of innovative structural systems, technological methods of shaping architectural images [5,15,20]. Dialogue with architectural images formed according to the properties and features of traditional tectonics allows the transition to new possibilities of display (in fact, “neotectonics” as a conductor of a new concept of displaying imagery) through the consistent elimination of the problems of the technological component of the utopian beginning of the compositional solution Neotectonics is considered in the context of cultural opposition and the way of conveying the peculiarities of experimental shaping through the expression of technological possibilities and the specifics of presenting a meaningful design of an architectural image [11,21,22,23]. It is the innovative methods of shaping and construction production technologies that remain the only and practical opportunity to form, restore or increase the parameters of the functional state of architectural objects, especially for special climatic and town-planning conditions or when forming unique or technologically complex architectural systems. The synergy of utopian and pragmatic principles for the aesthetic and rational solution of the tasks of architectural creativity is becoming a characteristic feature of the implementation of innovative technologies carried out within the framework of the stylistic direction of technotopia. 4 E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124405015 The evolutionary development of architectural science is increasingly focused on the practical implementation of the strategy of “collision and interpretation of differences”, using the novelty and diversity of technological possibilities. Technological adaptation of the achievements of various fields of knowledge in architectural creativity allows effectively solving problems associated with increasing durability and safety, and also expands the possibilities of compositional, constructive and functional transformation of architectural space. 3 Results The analysis of the fundamental components (utopian and pragmatic principles) of architectural creativity is carried out. The structure of signs of an architectural utopia has been developed. The significance and algorithm of interaction of the utopian and pragmatic principles for the evolutionary development of architectural activity are revealed. The mechanism of implementation of the possibilities of the pragmatic principle, realized in the format of the figurative and stylistic direction of technotopia, into architectural creativity is considered. The possibility of expanding the traditional areas of application and methods of architectural creativity in adapting technological advances from various fields of knowledge is shown. 4 Discussion In the general composition of modern concepts and the latest projects, it is rather difficult to single out the most significant results of architectural activity due to the lack of the necessary time distance. In this regard, it is permissible to analyze promising research objects as certain results that evolved from previous searches for utopia and technological methods of its practical implementation. The utopian assessment of objects and phenomena continues to be an internal objective property or measurement of human consciousness. Therefore, the main direction of development should be considered the formation of conditions for versatile harmonious interaction with utopia instead of living according to utopia. References 5 E3S Web of Conferences 244, 05015 (2021) EMMFT-2020 https://doi.org/10.1051/e3sconf/202124405015 2. T.A. Pchelintseva, Functions of utopia. Patterns of development of modern science: Collection of articles (Tomsk, 1981) 3. O.A. Chepelik, Art magazine 51/52, 67−69 (2003) 3. O.A. Chepelik, Art magazine 51/52, 67−69 (2003) 4. A.A. Musatov, The architecture of ancient Greece and ancient Rome (Architecture M., 2006) 5. E.E. Biryukova, Aesthetics of the form and content of the architectural space: dissertation for the degree of Ph.D. in Philosophy: 09.00.04 (Vladimir, 2003) 6. R. Klanten, L. Feireiss, Utopia Forever: Visions of Architecture and Urbanism (Die Gestalten Verlag, Berlin, 2011) 7. B. Goodwin, The Philosophy of Utopia (Routledge, London, 2001) 8. N. Coleman, Utopias and Architecture (Routledge, London, 2005) 9. R. Fishman, Urban Utopias in the Twentieth Century (MIT-Press, New York, 1982) 10. A.I. Khomyakov, Academia. Architecture and Creativity 3, 69−75 (2017) 11. C. Abel, Architecture, Technology and Process (Architectural Press, London, 2004) 12. F.D. Scott, Architecture or Techno-utopia: Politics after Modernism (The MIT Press, New York, 2010) 13. R. Klanten, The Tale of Tomorrow. Utopian Architecture in the Modernist Realm (Die Gestalten Verlag, Berlin, 2016) 14. P.S. Kapustin, Development of ideas about the design object in the processes of architectural thinking: dissertation for the degree of Ph.D. in Architecture: 18.00.01 (Voronezh, 1999) 15. I.A. Dobritsyna, From postmodernism to nonlinear architecture: Architecture in the context of modern philosophy and science: dissertation for the degree of Doctor of Architecture: 18.00.01 (M., 2007) 16. Statoil Regional and International Offices, https://www.archdaily.com/359599/statoil- regional-and-international-offices-a-lab 17. ING House/MVSA Architects, https://www.archdaily.com/568127/ing-house-mvsa- architects 18. AD Classics: Kubuswoningen/Piet Blom, https://www.archdaily.com/482339/ad- classics-kubuswoningen-piet-blom 19. Vodafone Headquarters/Barbosa & Guimarães, https://www.archdaily.com/54336/vodafone-headquarters-barbosa-guimaraes 20. Yu.I. Likhansky, Metaphors of the techno world in the architecture of the XX-t century (FESTU Publishing House, Vladivostok, 1999) 21. E.A. Lapshina, A.Yu. Likhansky, Vestnik of the FEFU Engineering School 2(27), 79- 90 (2016) 22. E. Anoshkina, E. Markovskaya, A. Mottaeva, As. Mottaeva, E3S Web of Conferences 210, 13022 (2020) https://doi.org/10.1051/e3sconf/202021013022 23. E.Yu. Shumilova, A.V. Tantsura, Collection of scientific reports of the 21st scientific and practical conference (North-Caucasian Branch of the Belgorod State Technological University named after V.G. Shukhov, Mineralnye Vody, 2015) 24. E. Ganebnykh, O. Lezhnina, J. Zhukova, V. Kashintseva, E3S Web of Conferences 210, 10008 (2020) 25. A. Mottaeva, A. 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https://openalex.org/W2074667750	https://zenodo.org/records/1949664/files/article.pdf	Italian	null	Sulle funzioni di green d’ordinem	Rendiconti del Circolo Matematico di Palermo	1,905	public-domain	14,838	Adunanza del x2 febbraio I9o 5. In questa Memoria studio in modo speciale le proprietk di certe funzioni poliarmoniche in un corpo circolare o sferico. Nei cap. I e II stabilisco per le funzioni poliarmoniche e per le funzioni di GREEN d'ordine m alcune propriet?t che sono una estensione di altre ben note relative alle funzioni armoniche e alla ordinaria fun- zione di GRE~.N. Nel cap. III considero un'area circolare e faccio vedere come certi integrali, estesi a tutto il cerchio, e relativi a prodotti di funzioni poliar- moniche per funzioni di GREEN d'ordine qualunque, possano esprimersi o sotto forma finita, o mediante integrali semplM (cio~ quadrature). Estendo poi queste proprietor al caso di un campo sferico. Per ottenere questi risultati non occorre affat.~o la conoscenza delle funzioni di GREEN. I1 cap. IV ~ destinato alia costruzione delie funzioni G, di GREEX d'ordine m e di i * specie per campi speciali. Esprimo perci6 la funzione G,, mediante funzioni poliarmoniche che determino successivamente con un metodo assai semplice. Tale funzione G m ha un segno costante nel campo considerato; inoltre essa si pu6 porte, anche nel caso di quante si vogliano variabili, sotto una forma assai semplice, mediante la consi- derazione di un integrale definito; questa forma si presta bene a stabilire un'altra proprietor, della funzione G,, che ~ l'estensione di una gi~t nora dovuta al POINCAR~. Nel cap. III considero un'area circolare e faccio vedere come certi integrali, estesi a tutto il cerchio, e relativi a prodotti di funzioni poliar- moniche per funzioni di GREEN d'ordine qualunque, possano esprimersi o sotto forma finita, o mediante integrali semplM (cio~ quadrature). Estendo poi queste proprietor al caso di un campo sferico. Per ottenere questi risultati non occorre affat.~o la conoscenza delle funzioni di GREEN. I1 cap. IV ~ destinato alia costruzione delie funzioni G, di GREEX d'ordine m e di i * specie per campi speciali. Esprimo perci6 la funzione G,, mediante funzioni poliarmoniche che determino successivamente con un metodo assai semplice. Tale funzione G m ha un segno costante nel campo considerato; inoltre essa si pu6 porte, anche nel caso di quante si vogliano variabili, sotto una forma assai semplice, mediante la consi- derazione di un integrale definito; questa forma si presta bene a stabilire un'altra proprietor, della funzione G,, che ~ l'estensione di una gi~t nora dovuta al POINCAR~. Ren& Circ. Maum..Palermo, t. XX (x9o5).--Stampato il z 9 maggio x9o5. Adunanza del x2 febbraio I9o 5. Dall'espressione delia funzione G,,, nel caso dell'area circolare, si deducono agevolmente dei limiti superiori per i massimi valori assoluti delle derivate dei primi 2m- I ordini di G,; questo ~ mostrato nel cap. V. Se ne trae poi di conseguenza che l'integrale u dell'equazione z 3 TOMMASO BOGGIO. 9 8 a2,, u = F (x, y) (F essendo una data funzione di x, y), che si annulla, suI contorno, colle sue derivate normali successive dei primi m -- I or- dini ~ finito colle sue derivate parziali dei primi 2 m -- I ordini, in ogni punto del cerchio e del suo contorno. Inoltre i massimi valori assoluti di queste derivate tendono a zero quando ii raggio del cerchio decresce indefinitamente. Dimostro infine le proposizioni analoghe nel caso di un campo sferico. Queste propriety, come mostrer6 in una prossima pubblicazione, trovano una applicazione immediata nella determinazione di una funzione u, regolare in un'area ~, semplicemente connessa, che soddisfa nei punti di a all'equazione l..~ami~--f(u) [ove f(u) ~ una funzione data di u e deUe sue derivate parziali dei primi 2 m -- 2 ordini], e che sul contorno assume colle sue derivate normali successive dei primi m -- I ordini, dei valori assegnati. I1 metodo che conviene adoperare ~ un'estensione di quello classico delle approssimazioni successive, proposto dal PICARD per integrate le equazioni di 2 ~ ordine della forma • =---f(u) [f(u) es- sendo una data funzione di u], colla condizione u = o sul contorno. ) Ricordiamo the una funzione u si dice m-armonica, o poliarmonica di ordine m, in un dato campo, se ~ regolare in tale campo e soddisfa al1'equazione A~m u = o. Generalizzazione delle formole di Green. I. Consideriamo un volume S limitato da una superficie chiusa ~; indichiamo con n la normale a ~ diretta all'interno di S e con u, v due funzioni regolari nello spazio S; allora si ha ovviamente, mediante inte- grazioni per patti ): / / ~t .... UA.sVdS+ fa2 ~ ud• .... V d'~ 9 /'[o~a=, ,uc~a2, 2v c3• .... uOa .... v } _ - dS--o, / . . . .... .... + .... .... + .... ,, .I, t ffdA2r 2 u / j, -~ a .... vd~+ ,t,~a .... vdS=o, ) Indichiamo, secondo l'uso, con A 4, A 6 .... i simboli operatori A 2 A2, A A A ..... ore A 2 --- ~-~ + ~-~ -{- ~-~ nel caso di 3 variabili, e • = ~'~ -[- ned r di 2 variabili. SULLE FUNZIONI DI GREEN D'ORDINE m. 99 onde sottraendo : onde sottraendo : /(4 .... .... A2~uA v) dS O) + -.--~ .... " dn • .... v----~n~ ]da = O; O) questa formola, del resto, discende subito da un noto lemma di GREEN. Indichiamo con m un numero pari, e poniamo in questa formola v -- u, poi successivamente : r-'- m, $-'-- I r=m~I, s --2 . . . . , . . . . ~ ~ . ~ ~ r-'- m, $-'-- I r=m~I, s --2 . . . . , . . . . ~ ~ . Generalizzazione delle formole di Green. ~ ~ e sommiamo le formole che cosl si ottengono; risulterA: A 2 [( .)-..]dS nella quale formola compariscono, sul contorno, i valori delle funzioni: nella quale formola compariscono, sul contorno, i valori delle funzioni: dA=u dA =u d~f, A2 ~, ~ 9 9 9 A2m_2 U ' -- (a) u, dn' dn ' ' dn (a) Supponiamo che su ~ siano nulle le prime m delle quantiul (a), allora risuha dalla (2): /ua2.udS --/(a=,u)~dS; se poi la funzione u ~ m armonica in S ), ne segue: A2pU ~- 0 in tutto il campo S se poi la funzione u ~ m armonica in S ), ne segue: se poi la funzione u ~ m armonica in S ), ne segue: A2pU ~- 0 in tutto il campo S in tutto il campo S Se poi m ~ un numero dispari e si pone v---- (2) si ha la seguente: , invece della 2 ) Ricordiamo the una funzione u si dice m-armonica, o poliarmonica di ordine m, in un dato campo, se ~ regolare in tale campo e soddisfa al1'equazione A~m u = o. T O M M A S O B O G G I O . I O 0 A 2 A 2 A 2 ~u ~ ~u uAmufdS * 1 __ v~ p/ da ,v ~i u dA ~+ ~iu \ p d• u \|da-- IA 2#-= -cl,--~o " ~u ~ ~u uAmufdS 1 __ v~ p/ da ,v ~i u dA ~+ ~iu \ p d• u \|da-- IA 2#-= -cl,--~o perci6 se su cr sono nulle le prime m delle quantitk (a) si deduce: perci6 se su cr sono nulle le prime m delle quantitk (a) si deduce ua,mud S_ _ 0 ~u [~ ,~u~ _L dS; .,, + !" se inoltre la funzione u ~ m-armonica in S, se ne trae: e inoltre la funzione u ~ m-armonica in S, se ne trae: ~0~ Ox - - Oy - - OZ delle quali segue facilmente, in ogni punto di S: delle quali segue facilmente, in ogni punto di S: &2v U --- 0. Cosl proseguendo, ~ facile dedurre, qualunque sia m, che la fun- zione u, poliarmonica d'ordine m in S, e della quale su ~ son nulle le prime m delle quantit~ (a), ~ identicamente nulla in ogni punto di S. #. Generalizzazione delle formole di Green. chiaro the se sopra ~ scno nulle le prime m delle quantit~ (a), saranno pure nulle, nei pumi di <r, le derivate normali dei primi m- I ordini di u, e viceversa; perci6 si pu6 anche dire chese una funzione u, m-armonica in S, si annulla sul contorno colle sue derivate normali dei i i I di i id i ll i i[ S primi m I ordini, sar identicamente nulla in tutto i[ campo S. Come conseguenza immediata si ha: Se di una fun~ione u, regolare in S, si conosce il ~,,, e se suI contorno ~ le prime m delle quantittt (a) assumono valori assegnati, Ia funz~ione u risulta completamente determinata in ogni punto di S. Ovvero: Una fun@ne u, regolare in S, di cui si co- nosce il • ~ completamente determinata nell'intero campo S, se sul con- torno ~ si conoscono i valori della funxione u e deIle sue derivate normaIi successive dei primi m -- I ordini. 2. Poniamo ora nella formola (1) successivamente: r---m~ s-- I r--- m-- I~ $-- 2 9 + 9 9 + 9 , 9 9 , 9 9 9 9 9 9 9 9 9 , + 9 . 9 9 9 9 9 r--'2, s--m-- I r-- I~ $---m SULLE FUNZIONI DI GREEN D'ORDINE m. I0I e sommiamo te formole che cosl risultano; si avrk; e sommiamo te formole che cosl risultano; si avrk; f(uA v--VamU)dS (3) -t.- ~ P/a uda .... ~v da ~ ~u'~ (3) che chiameremo iI Iemma di GREEN per I'operaKione • Se, in particolare, le funzioni u, v sono m-armoniche in S, si ha: che chiameremo iI Iemma di GREEN per I'operaKione • che chiameremo iI Iemma di GREEN per I'operaKione • che chiameremo iI Iemma di GREEN per I'operaKione • Se, in particolare, le funzioni u, v sono m-armoniche in S, si ha: p p Se, in particolare, le funzioni u, v sono m-armoniche in S, si ha: Se, in particolare, le funzioni u, v sono m-armonich ~f~(A d• ..... iv dA~i_~U)d~.__ ~ ; ~i-~ u d n A .... i v d n ' questa formola pub ancora scriversi: (39 \ a v ] =o. Poniamo ora : Poniamo ora : ~ rarn-3 over ~ Ia distanza dell'origine O delle coordinate da un punto qualunque (x, y, Z). ) Questa formola, per m ~-2, 6 stata data dal MATHIEU nel suo lavoro: M~- Generalizzazione delle formole di Green. Se il punto O 6 fuori di S, la funzione v, data dalla formola precedente, e regolare hello spazio 5, quindi la formola (3) sussiste inalterata. Se invece l'origine ~ un punto di S, a distanza finita dal contorno, le derivate di v dell'ordine 2 m -- 2 diventano infinite precisamente in questo punto, quindi la (3) non sussiste pi/1. Escludiamo perci6 il punto O da S mediante una sfera S o, di centro l'origine O, di contorno %, e di raggio Ro piccolo in modo che questa sfera sia tutta contenuta nel campo S. La funzione r ='-s risulta allora regolare nello spazio S- S o, ed inoltre, come 6 facile verificare, ~ m-armonica in tale spazio; perci6 la formola (3) applicata alle funzioni u, r ""-3 che sono regolari nelIo spazio S- S o, il quale ha per contorno ~ + %, porge: ~ fs- r~m-3A~mudS $o Aai_ 2 ~ d A2m_2 i r 2m.-3 i ~'o d gr A2rn-2i ?" d n od ancora : od ancora : od ancora : ) Cfr. GUTZMER, Remarques sur certaines ~quations aux diff&ences partielles d'ordre sup&ieur [Journal de Math6matiques, IV c s6rie, t. VI (x89o), pp. 4o5-422]. TOMM&SO BOGOIO. I02 fs-so r~'-~ A,. u d S "--" i A21__2 U 2m-2idn r A2 m - 21 f2m--3 tin-- d 6" + ~ d n o .... ~ d n o ore n o ~ la normale a .o diretta all'esterno di S o. Si faccia ora tendere a zero il raggio Ro, aUora ~ facile vedere che l'ultima ~-- tende a zero; e osservando che ~'-3 (2 m -- 231 ~ A2m_2 r --- -" r ~'-3 (2 m -- 231 ~ A2m_2 r --- -" r si otterrA: ;( ' J:o) 21 - (2m--2) llim u . de Ro=~ t./tr \ ~n~no r si otterrA: ;( ' J:o) 21 - (2m--2) llim u . moire sur l'dquation aux diffdrences, etc. [Journal de Math~matiques, IIe s~rie, t. XIV (I869), pp. 378-42I]. Per m qualunque, l'ho data (senza dimostrazione) nella mia Nota : Un teorema di reciprocitd sulle funzioni di GREEN d'ordine qualunque [Atfi Acc. Torino, vol. XXXV 09oo), pp. 498-5o9]. Generalizzazione delle formole di Green. de Ro=~ t./tr \ ~n~no r ;( J ) Chiamiamo inoltre u oil valore di u he1 punto O, ricordiamo che d%-~-R~o senOdOd% e si avrA: r I du de-- senOdOd~ U dn o r dn o/ -- u~ go go __ (u -- Uo) senOdOd~ -d-~n RosenOdOdg, r I du de-- senOdOd~ U dn o r dn o/ -- u~ go go __ (u -- Uo) senOdOd~ -d-~n RosenOdOdg, onde : r I du d% ~m u dno/ -- -- 4~Uo, -%-otj= k dn o r quindi infine : r,, ~" "A 2m-3 a 2m_ulr -a+,~_+,r 2m idA21 2 U ' - dn )d (+) f + + + dS onde : quindi infine : r,, ~" "A 2m-3 a 2m_ulr -a+,~_+,r 2m idA21 2 U ' - dn )de (+) -- fsr+•-+ a+,.udS, (+) che si pu6 chiamare la formola di GREE~ per l'operazione A.,. ). che si pu6 chiamare la formola di GREE~ per l'operazione A.,. ) Questa formola, per m ~-2, 6 stata data dal MATHIEU nel suo lavoro: M~- SULLE FUNZIONI DI GREEN D'ORDINE m. IO3 Seil punto 0 cade su~ basra porre nel primo membro di questa formola 2 ~ al post 0 di 4 ~. Questa formola vale anche se il campo S 6 lo spazio indefinito esterno ad una o pia superficie chiuse; bisogna per6 supporre in tal caso che quando r tende ad o% le derivate d'ordine i delia funzione u moki- plicate per /§ (i = o, i, ..., 2 m -- I) tendano verso limiti determi- nati e finiti. Per funzioni di 2 variabili, definite in un'area piana ~, limitata da un contorno chiuso s, bisogna invece porte: v = r ~m-~ log r, e si ottiene la formola corrispondente ddla (4): i"[" d,.Xm ~(r_~n .... logr)__ ~m_~,( r logr)___d_~__n ~dS (4t) ..I --2 ..... (logr)A mud.. Le funzioni di Green d'ordine m e loro proprieth generali. Le funzioni di Green d'ordine m e loro proprieth generali. 3. Chiameremo funzjone preliminare di GREE~ d'ordine m e di I ~ specie la funzione I'm, m-armonica in S, e che su , soddisfa alle condizioni: d~ rm d~ r2"-3 (5) -- - - , Ci = o, ..... m- x), d n ~ d n ~ d~ rm d~ r2"-3 -- - - , Ci = o, ..... m- x), d n ~ d n ~ (5) ore r ~ la distanza di un punto fisso qualunque M di S da un punto mobile. ore r ~ la distanza di un punto fisso qualunque M di S da un punto mobile. Si vede facilmente, analogamente a quanto si ~ detto in fine del ~ I, che essendo verificate le (5), saranno soddisfatte le seguenti sem ~ pari: l ( ) ~21--2 I'm --"- A21-2 m (6) dA2i_2r m dA~i_~r ~-3 i= i, 2 ..... ~ , dn dn l ( ) ~21--2 I'm --"- A21-2 m dA2i_2r m dA~i_~r ~-3 i= i, 2 ..... ~ , dn dn (6) moire sur l'dquation aux diffdrences, etc. [Journal de Math~matiques, IIe s~rie, t. XIV (I869), pp. 378-42I]. Per m qualunque, l'ho data (senza dimostrazione) nella mia Nota : Un teorema di reciprocitd sulle funzioni di GREEN d'ordine qualunque [Atfi Acc. Torino, vol. XXXV 09oo), pp. 498-5o9]. TOMMASO BOGGIO. 104 ovvero queste altre se m 6 dispari: ovvero queste altre se m 6 dispari: ovvero queste altre se m 6 dispari: A~i_~ Fm -- A2i_:r~m-3 (6') dA2/_2 F m dA21_2rZm-3( m--I) dn -- dn i=i, 2 ..... Am_ ~ F m ~- Am_ ~ r 2m-3 (6') e queste condizioni possono sostituirsi aUe (5). Se si pone: e queste condizioni possono sostituirsi aUe (5). Se si pone: Gm -- r2"-; -- rm, Gm -- r2"-; -- rm, Gm -- r2"-; -- rm, (7) ( ) ; , la funzione G,. ~ nulla su ~ colle sue derivate normali successive dei primi m- I ordini; + poi m-armonica nel campo S salvo nel polo M, da cui si contano le distanze r, perch6 in questo punto le derivate par- I ziali d'ordine 2 m- 2 diventano infinite come --. Y Y La funzione G la chiameremo fun~ione di GREEN d'ordine m e di I a specie ). ) Nel caso di due variabili, la funzione G= ~ suscettibile di aria semplice inter- pretazione fisica. Cfr. in proposito la mia Nota: Sull'equilibrio delle piastre elastiche imastrate [Rend. Acc. Lincei, vol. X, I ~ sem. 19oi, pp. x97-2o5]. Le funzioni di Green d'ordine m e loro proprieth generali. In qualche lavoro precedente ho chiamato funzione di GREE~ d'or- dine m e di i ~ specie la funzione Fm; ora invece, segue'ndo il POI~CAR~ ed altri autori, ho indicato con tale nome la funzione G,., che, del resto, ~: queUa che comparisce nella maggior parte delle formole che incon- treremo; poich~ per6 occorre talvolta considerate la funzidne Fro, cosl, come dissi, verr~ chiamata funzione preliminare di GREE~ d'ordine m e di I* specie. t~ chiaro che la funzione G,., al pari dig,, dipende soltanto dalla forma del contorno o, ed ~ funzione di due terne di variabili. Nel ~ I si 6 dimostrato che una funzione u, regolare in S, di cui 6 noto il A,,,, ~ determinata in ogni punto M di questo campo allorch+ sul contorno si r i valori delle prime m delle quantit.~ (a); ora mostreremo che se si sa determinare, nel campo S, la funzione di GREV.N d'ordine m e di I" specie, il cui polo 6 il punto M, si saprl pure tro- vare il valore, nel punto M, della funzione u. Infatti, chiamando u(M) tale valore, la (4) porge: t (2m__2)!4~u(M) A c. (49 /, .... ,a , ..... ,,, 9 ]"-Js" t (2m__2)!4~u(M) A c. (49 /, .... ,a , ..... ,,, 9 ]"-Js" (49 ) Nel caso di due variabili, la funzione G= ~ suscettibile di aria semplice inter- pretazione fisica. Cfr. in proposito la mia Nota: Sull'equilibrio delle piastre elastiche imastrate [Rend. Acc. Lincei, vol. X, I ~ sem. 19oi, pp. x97-2o5]. SULLE FUNZIONI DI GREEN D'ORDINE rtl. IO 5 ma dalla (3) appllcata alle funzioni Pmed u si ottiene: - ] ' onde sottraendo e ricordando la(7) e le (6) se m ~ pari si ha: onde sottraendo e ricordando la(7) e le (6) se m ~ pari si ha (8) --/GmA==udS (8) la quale formola, supposta nota la funzione G,,, fornisce appunto il valore della u nel punto M. Re~d. Clrr Maum. Palermo, t. XX (xgoi).- Stampato il x 9 maggio 19o 5. ) Per m = 2 questa formola ~ stata data dal Prof. LAURICELLA nella sua Me- moria: Sull'equatione delle vibra~ioni dells placche elastiche incastrate [Memorie della R. Accademia ddle Scienze di Torino, serie II, t. XLVI (I896)]. Le funzioni di Green d'ordine m e loro proprieth generali. Se m ~ dispari, invece ddle condizioni (6)bisogna applicare le (6') e si ottiene cosi: ku(M) (8') dn [ m i] k= (2m-- 2)14~; "~ ~-----7- " (8') In particolare, indichiamo con F(x, y, :0 una funzione data, continua colle sue derivate di I ~ ordine, e cerchiamo la funzione u, regolare in S, che soddisfa ivi aU'equazione : e nei punti del contorno * aUe altre: e nei punti del contorno * aUe altre: d i u -- O, (i=O, I ..... m--I). d n i Da queste condizioni e da quanto si disse nel ~ I, risutta che nei punfi di , si avr~t pure: Da queste condizioni e da quanto si disse nel ~ I, risutta che nei punfi di , si avr~t pure: A21 U ~ O~ ( dn -"o i~o, I, ..., sem ~ pari; ovvero: d • u A2iqd. ~ O, d?l = O~ m~3) An_ U~O i=o, I ..... -~ ~4 Re~d. Clrr Maum. Palermo, t. XX (xgoi).- Stampato il x 9 maggio 19o 5. Re~d. Clrr Maum. Palermo, t. XX (xgoi).- Stampato il x 9 maggio 19o 5. ~4 ~4 TOMMASO BOGG. I O. IO6 se m 6 dispari. Perci6 dalla (8) od (8') si deduce in ogni caso: se m 6 dispari. Perci6 dalla (8) od (8') si deduce in ogni caso (9) k U (X, y, ~.) = --~Gm(x , y, 7,.; ~, "~, ~)F@, ~, ~)dS (dS = d~d'~d~). Per funzioni di 2 variabili bisogna sostituire in luogo di r ..... 3 l'espres- sione r .... log r, perci6 invece delle (~), (7) avremo: d ~ r m __ d i (r .... log ~;) (i = o, ~ ..... m -- O, (5,) d d n (7,) Gm = r ~"-~ log'r -- r., ; (5,) (7,) applicando poi la (4,) si ottiene la formola seguente, analoga alla (8): k' u(M) ~ dn - dn Z -- OmA2m"da ) k' u(M) ~ dn - dn Z (8,) -- OmA2m"da ) (8,) ed un'altra formola, che non scriveremo, analoga alia (8'). ed un'altra formola, che non scriveremo, analoga alia (8'). ed un altra formola, che non scriveremo, analo La formola corrispondente delia (9) e: La formola corrispondente delia (9) e: k'u(x, y) = /'Ore(X, y; ~) F (~ ~)dq. (9,) 4. Le funzioni di Green d'ordine m e loro proprieth generali. Consideriamo una funzione u, regolare in S, di cui sia noto in S il a2m, e sul contorno i valori deile quanfifft : dA u d A2s 4 U d A25?~__I ~ (b) u, dn ' A~'3u' dn '"" dn ' a~'mU' dA u d A2s 4 U d A25?~__I ~ (b) u, dn ' A~'3u' dn '"" dn ' a~'mU' (b) ore s~, s3, ... s .... , s m sono numeri interi e positivi (dei quali il primo soltanto pu6 esser zero) e minori di m, disposti per ordine di grandezza crescente e tali inoltre che tre consecutivi non siano mai eguali; nei ter- mini (b) compariscono delle derivate i cui ordini sono rispettivamente O, 2S 2 -+- I, 2S3, ... , 2Sin_ , "3 l- I, 2S/; orbene supporremo ancora che questi numeri siano tali chela somma di due qualunque di essi non sia mai eguale a 2m -- I. Per determinate la funzione u, allorquando sul contorno si cono- SULLE FUNZIONI DI GREEN D'ORDINE m. SULLE FUNZIONI DI GREEN D'ORDINE m. lO 7 scono i valori deUe quantit5 (b), basra determinare una conveniente fun- zione di GREES d'ordine m. Consideriamo infatti una funzione r'm, m-armonica in S, e che nei punti di ~ verifichi le equazioni: r' -- r am., tn r' T 2m-3 dn -- dn A r' =~ r 2"-3 2s 3 m ts 3 0o) . . . . . . . . . . . . . . . d • F' r ~-3 ,, __ d • .... dn dn r'~. r 2m-3 A2s m ~ A2s m ; 0o) sottraendo aliora la (3"), nella quale si legga r' al posto di r+, dalla (4'), ricordando le (IO), e ponendo, analogamente alla (7): G' ~ r 2~-3 -- F' facile ottenere : I d• .... G'.dr t~d• u , ku(M) d n --# ~ -'-d~n ..... 4--2 + ; d ,X ...... -~ " d 9 .. 2 c- a2Sr~ u d-n ~ -- G'mA2mudS' Js la quale dimostra la propriet.'i enunciata. 1~ chiaro che la funzione r' ora considerata, dipende dalle coordi. hate del polo e del punto mobile; a questo riguardo ora dimostreremo che ~ simmetrica rispetto a queste due terne di coordinate ). ) Questo teorema 1o diedi, senza dimostrazione, nella mia Nora gi~ citata: Un teorema di re@rocitG etc. Le funzioni di Green d'ordine m e loro proprieth generali. Siano perci6 Po(xo, Yo, G), P, (x, y,, ~,) due punti di 5; indi- chiamo con r'm(Po, P3 il valore in Po della funzione F' m avente per polo il punto P, e con F',,(P, Po) il valore in P, della funzione r' mil cui polo ~ Po; allora si vuol dimostrare che: r' (Po, P,) = r'm (P,, Po). I08 TOMMASO BOGGIO. Se P(x, y, Z) e un terzo punto di S e si pone: Se P(x, y, Z) e un terzo punto di S e si pone: ro (P) = r'm (P, Po) r (P) = p', (P, P), ro (P) = r'm (P, Po) r (P) = p', (P, P), ro(2,) = r, (&). baster~t far vedere che Chiamiamo perci6 to, r, le distanze di Poe P, da P; dalla (II) si ha, ponendo per G'~, la sua espressione: k Fo(P,) --- Po dn dn f d A r o -- d----7--t (a ..... =_=rT"-' -- a ..... =_= r)d. + ..- ..... =_,,= d a/,,_~,,,_= F, d ~, "'" -{- A2ra r~ dn dn d • r o kr (Po) = r dn dn _ f dA= P, j, Tn (a= .... =-= r:"-' -- a ..... =-, ro) d, + ... 9 . 9 + a=, m r, - ="-~-~=- . . . . . . . -dn-= Sottraendo si deduce : Sottraendo si deduce : p/d• r, - A f~( A 2'n--3 d 2+fill__ 2 $2__2 r 0 ""+ G'~ r~ dn (d~,\ o dn l"[da,~ r o 2m-- 3 _ ~,,'-3 dam_ 2 rx ,~ d• = r o po dz d a= I" o "-3'~ d----7--a,,,_,a_,r, )da + ... d a r ="-3 \ ..... "- ' \| d --A,, ro ~ ) d A"-" F~ ) de F, I" d n A Po] d Consideriamo ora gli .~ contenuti tra le { ... }; essi sono m in tutto, inoltre, per le ipotesi fatte sui humeri s, ~ facile riconoscere the questi SULLE FUNZIONI DI GREEN D~ORDINE m. IO 9 SULLE FUNZIONI DI GREEN D~ORDINE m. IO 9 integrali sono tutti diversi tra loro e che quindi essi non sono altro che quelli contenuti neU'espressione A =.~._=, A= i pd _ do ~[[i =/-2 F~ d-n - dn scritti per6 in ordine differente; ora, in virtfl della (3') l'espressione pre- cedente vale zero. A =.~._=, A= i pd _ do ~[[i =/-2 F~ d-n - dn A =.~._=, A= i pd _ do ~[[i =/-2 F~ d-n - dn scritti per6 in ordine differente; ora, in virtfl della (3') l'espressione pre- cedente vale zero. Tenendo conto deUe (io), l'eguaglianza precedente diventa: d~ ~ ,j ~, \ d n • r~'~-3o ~'=d n~ • _,,~_= , /do_jr - ... =m-3 -- r=o "-3 d A ..... m--a r~ =-3) --.t"[a =m_ g" ...... _=ro a do, +A\ d. Se P(x, y, Z) e un terzo punto di S e si pone: "m a, g 9 B / e per quanto si disse or ora possiamo anche scrivere: k [r (R) -- ro &,)] d a.._=/ m--3 d = r, d __ am-- 3 ro - _2m--t -- i Aal-2r d ~ -- 'xai--2r~ ---- G, ed ancora : ed ancora : k [r (Po) - ro (e,)] -- --+o.Z- _l(A, =,_= r,="-3 d • r;"-3d n A ~,. _ ~, ro'~ dA " r ~'-~n = .... .)do;'\ mail 2 ~ membro vale zero, come ho mostrato nel ~ 4 della mia prima Nota citata, onde si conclude: mail 2 ~ membro vale zero, come ho mostrato nel ~ 4 della mia prima Nota citata, onde si conclude: r, (Po) = ro (P,), come volevasi dimostrare. come volevasi dimostrare. NeUa mia Nota gi~t citata: SulI'equilibrio delle piastre, ecc., ho fatto vedere che questo teorema di reciprocitk, nel caso di 2 variabili e della funzione G=, dk luogo alHnterpretazione fisica seguente: Sia a una piastra elastica, piana, disposta orizzontalmente, e sia applicato in un punto qualunque .4 di essa un peso P; aUora lo spostamento (verticale) in un aitro punto qualunque M di ~ ~ lo stesso di quello che si avrebbe nel punto A se il peso P fosse applicato in M. Un caso assai parficolare di questo teorema, e precisamente il caso in cui la piastra ~ ~ circolare eil punto M ~ nel centro, trovasi a pag. 138 dell'opera del BoussI~.so_, Applications des potentiels h l'gtude de l'dquilibre et du mouvement des so- lides glastiques (:Paris, 1885). TOMMASO BOGGIO. II0 retativi alte funzioni di Green d' ordine m. 5. Indichiamo con ~ un'area circolare di raggio R, il cui contorno verrA indicato con s; come origine delle coordinate assumeremo il cen- tro O di ~. Denotiamo poi con F(x, y) una funzione data, n-armonica in ,; aUora la funzione u, regolare in ~, che soddisfa alle equazioni: (I2) • u=F (in .) 03) . = o (su ,) 03) 6 data dalla formola (9,) del w 3 ove vi si faccia m "--I, e si ha Cosi: (I4) 2~u(x, y) = G~(x, y; ~, ~)F(~, ~)d~, (I4) ove G, 6 l'ordinaria funzione di GREEN. ove G, 6 l'ordinaria funzione di GREEN. Questa formola esprime la funzione u mediante un integrale dop- pio; ~ per6 facile trovarrie un'altra che dia la u espressa per mezzo di integrali semplici (quadrature). Osserviamo per questo che la funzione n-armonica F pu6 rappre- sentarsi, come ~ ben noto, colla formola : n--I ~o 21 X 2 (15) F(x, y) -= , ? f,, (~ = +/), (15) le fl essendo funzioni armoniche conosciute. le fl essendo funzioni armoniche conosciute. Sostituendo nella (i2) si ha: Sostituendo nella (i2) si ha: n--i . 06) • u -- o~ , ?~'f,, 06) la quale mostra che la funzione u a poiiarmonica d'ordine n + I, e pub perci6 scriversi: la quale mostra che la funzione u a poiiarmonica d'ordine n + I, e pub perci6 scriversi: pub perci6 scriversi: n 07) "= 07) le % essendo funzioni armoniche da determinarsi. le % essendo funzioni armoniche da determinarsi. Da questa eguaglianza si deduce: ( Da questa eguaglianza si deduce: ( -( ~Xu ~ 4s~? .... ~?s+ 4s? ~'-~ 09,\ SULLE FUNZIONI DI GREEN D'ORDINE m. I I1 e confrontando colla (t6): e confrontando colla (t6): e confrontando colla (t6): ~,p .... (4s~ ? --t- 4s,~-~-- f,_,) =o; ora, l'espressione entro parentesi ~ una funzione armonica, perci6 af- finch6 quest'equazione possa sussistere debbono necessariamente essere nulle le espressioni entro le parentesi, cio6: I 'r, + - 45 L-,' (S: I, 2, ... , ~/,), da quest'equazione si ricava con una nora formola : da quest'equazione si ricava con una nora formola : I s foil (,8) C'L_,a (,=,, ..... .3, (,8) (,=,, ..... .3, e cosl conosciamo gi~l le funzioni armoniche ~,, %, ..., q~ e cosl conosciamo gi~l le funzioni armoniche ~,, %, ..., q~. Rimane ora da determinare la funzione armonica %. 6. Indichiamo ancora con F(x, y) una funzione n-armonica data e cerchiamo la funzione u, regolare in , e che soddisfa age equazioni: retativi alte funzioni di Green d' ordine m. Dobbiamo perci6 ricorrere alia (I3) che ci d~i, tenendo conto della (17): della (17): ~o' R2' % = o (sopra s), e quest'equazione, com'~ chiaro, sussiste non solo nei punti di s, ma nell'intero cerchio ~. nell'intero cerchio ~. Se ne trae: n la funzione % ~ cosi conosciuta, e sostituendo neUa (t7) potremo scri- vere : n (I9) u= ~+(p2,__ R2,)q) , (I9) le funzioni % essendo date dalle (I8). le funzioni % essendo date dalle (I8). Questa formola esprime appunto la funzione u mediante integrali semplici. Se F ~ un polinomio, ~ pure, di conseguenza, una funzione po- liarmonica ; aliora neila (I5) le funzioni f~ sono polinomi armonici, per- ci6 dalle (tS) risulta che le q~ sono anch'esse polinomi armonici, e in- fine la funzione u data dalla (i9) sar~ pure un polinomio. 6. Indichiamo ancora con F(x, y) una funzione n-armonica data e cerchiamo la funzione u, regolare in , e che soddisfa age equazioni: TOMMASO BOGGIO, I12 (20) %.u =F (in .) d u dm-' u -- -- .... tin,,,-------- ~ -- o (su s). (2I) ~ = d (20) (2I) t~ chiaro intanto che la s u ~ poiiarmonica d'ordine m-}-n, perci6, come ~ noto, pu6 rappresentarsi con m 2 V n s armoniche ?o, ~?,, ..., 9 ..... mediame la s : m-+-n--i ~ - ~o. r ~'" m-+-n--i ~ - ~o. r ~'" e: a u-- ~,4s? .... s?,+p a scriversi: ..... 9 .-.r , Se ne trae: che pu6 ancora scriversi: che pu6 ancora scriversi: ..... 9 .-.r , ne viene, in modo analogo : ne viene, in modo analogo : ..... [. \ ~ (r A,u "--" ~2 ' 4"(S -- I)S~2s--4 "ps--2 I' e in generale : ..... , ._.r ~ a'(P~,/-I A U-"- ~, 4"(s--mGi-i)(s--m-Ji-21...(s--I)sp LF-" J ..... retativi alte funzioni di Green d' ordine m. , ._.r ~ a'(P~,/-I A U-"- ~, 4"(s--mGi-i)(s--m-Ji-21...(s--I)sp LF-" J Questa eguaglianza pub ancora scriversi: ore si ~ posto, per brevit',t: ore si ~ posto, per brevit',t: b, -- 4"(s-- m Jr- I)(S -- m + 2) ..- (s -- z)s; perci6 sostituendo nella (201 e ricordando l'espressione (151 di F b, -- 4"(s-- m Jr- I)(S -- m + 2) ..- (s -- z)s; b, -- 4"(s-- m Jr- I)(S -- m + 2) ..- (s -- z)s; perci6 sostituendo nella (201 e ricordando l'espressione (151 di F, si ha: perci6 sostituendo nella (201 e ricordando l'espressione (151 di F, si ha: "-~o ~,e''E I a" (P'+'~P'+'lh p, ap~, f,] -- o, dalla quale si deduce, come nel ~ precedente: dalla quale si deduce, come nel ~ precedente: I ara (~m+s ~pm._sl b, ~, a?" f,--o cio6 : a'(pm+' ~p,,+,) (23) at~r- -- -~ t~'f, 9 I ara (~m+s ~pm._sl b, ~, a?" f,--o I ara (~m+s ~pm._sl b, ~, a?" f,--o a'(pm+' ~p,,+,) at r 'f 9 cio6 : cio6 : a'(pm+' ~p,,+,) (23) at~r- -- -~ t~'f, 9 (23) Da quest'equazione si ricava: a'-' (P'+' %~-')a?'-' _-- ~7 f%'f, d?, SULLE FUNZIONI DI GREEN D~ORDINE m. I I quindi : ac = -sT, Z f, aP alp, e con una integrazione per pard: e con una integrazione per pard: ap,._~ =~ p pf, dp-- p ; ne segue : m+s - P " " P s 3p m-~ -- h, ed integrando per patti: ap,._~ =~ p pf, dp-- p ; ap,._~ =~ p pf, dp-- p ; ne segue : m+s - P " " P s 3p m-~ -- h, ne segue : ne segue : m+s - P " " P s 3p m-~ -- h, ed integrando per patti: ed integrando per patti: 3m-'(Pm+S~m+') I [ / p /Pp'+'f d /P ] c~p m-3 -- 2!h, p2 Cfdp -- 2? p -~- r dp ; cosl proseguendo si trova infine: cosl proseguendo si trova infine: Pm+'?m+' --(m- ? .... i.~o ?'+if, dr ; del resto 8 assai facile verificare che questa funzione soddisf~ effettiva- mente alia (23). Se ne deduce: I m--, __ i~p_,_~_~f%,+ifde (24) ?m+. -- (m __ l)! h[ ~o ;(- xf(m i ] do (s=O, I, ..., n--I), e cosl conosciamo le funzioni armoniche ?m, %+,, "'" , ? ...... 9 7. Rend. Circ. Mattm. Palermo, t. XX (19o5).- Stampato il 19 maggio ,905. ) BOGGIO, Sopra alcune funzion/ armoniche o biarmoniche, etc. [Atti del R. Isti- tuto Veneto di Scienze, Lettere ed Arti, 1:. LX, parte 2 a (19Ol)]. retativi alte funzioni di Green d' ordine m. Dobbiamo ora determinare le funzioni armoniche ?o, 7i, ... , %-,, e perci6 ricorreremo alle (21), che sinora non sono state adoperate. La prima delle (20, tenendo conto della (22), porge nei punti di s: m-t-,t-- 1 (25,) ~, R ~' ?, = o, (25,) e quest'equazione ~ valida non solo su s, ma in tutto il cerchio ,. DaUa (22) e dalla seconda delle (2I) si ha poi: clog poich~ p = R : __1( Rend. Circ. Mattm. Palermo, t. XX (19o5).- Stampato il 19 maggio ,905. I5 TOMMASO BOGGIO. II 4 e quest'equazione, come si riconosce subito, sussiste non solo nei punti di s, ma nell'intero cerchio . Dalla (25,) risulta poi subito che l'equazione precedente si riduce alia : alia : m-4-,l~ i (25=) ~, s R .... % -- o. (25=) Similmente, tenendo conto delle (25,), (25~) ~ facile vedere chela terza delle (21) conduce all'equazione: m-+-n-- , Z, ,(,- = o; e cosl procedendo si trova infine che l'uitima delle (21) fornisce l'e- quazione : e cosl procedendo si trova infine che l'uitima delle (21) fornisce l'e- quazione : m+~t--i y s(,-I)...(,-m+z)R ..... m--i Da quest'equazione si ricava q~ .... che ~ l'unica funzione incognita che vi figura; da queIla chela precede si ottiene poi q~,,_=, e cosl via; la (253) fornisce poi la funzione %, la (252) la funzione c?,, e la (25,) la funzione %. In tal modo tutte le funzioni armoniche ,% che figurano nella (22) sono conosciute e risultano espresse mediante integrali semplici (qua- drature). ) Ricordando poi la (9,) e le (I5), (22) potremo scrivere: Se la funzione F ~ un polinomio, le funzioni fi che compariscono nella (15) sono, come giA 6 stato detto, dei polinomi armonici, e allora le funzioni o date dalle (24), (25,) ... (25,,) risultano pure polinomi armonici, e in~ne la funzione u data dalla (22) sara anch'essa un po- linomio. Questo risultato vale ancora se il contorno s, invece the una cir- conferenza, ~ un'ellisse qualunque. Infatti sia Fun polinomio di un grado qualunque n; la funzione u che soddisfa aUa (20) hell'area limitata dall'ellisse considerata, e aUe (2I) sul contorno, ~ aUora un polinomio di grado 2m + n, come ho SULLE FUNZIONI DI GREEN D'ORDINE m. I I5 facto vedere in una Nota precedente ); esso si determina facilmente col metodo dei coefficienti indeterminati. retativi alte funzioni di Green d' ordine m. Sia cio~ : Sia cio~ : X2 y2 a--V+ b~ I --o l'equazione dell'ellisse s, e poniamo: ( l'equazione dell'ellisse s, e poniamo: ( (x y .= -~+ b~ ~ x(x,y), ore X a un polinomio di grado n di cui dobbiamo cercare icoefficienti. t~ chiaro allora che le (2I) sono soddisfatte ; sosfituendo poi nella (2o) e confrontando fra loro i due membri si trovano tante equazioni (effet- tivamente compatibili, cio~, come ~ assai facile vedere, col determinante dei coefficienti non nullo) quante occorrono per trovare tutti i co.efficienti del polinomio ;q che c0sl risulta conosciuto. ore X a un polinomio di grado n di cui dobbiamo cercare icoefficienti. t~ chiaro allora che le (2I) sono soddisfatte ; sosfituendo poi nella (2o) e confrontando fra loro i due membri si trovano tante equazioni (effet- tivamente compatibili, cio~, come ~ assai facile vedere, col determinante dei coefficienti non nullo) quante occorrono per trovare tutti i co.efficienti del polinomio ;q che c0sl risulta conosciuto. Se si suppone F = I ~ facile avere dalle (2o), (21) nel cerchio ~ : I a 2) '/~ -- R m 2 ~'~ (m!) ~ (~ -- onde, confrontando coUa (93: (26') d ~ 2 m ~ (~o -- R~) m , che ~ un caso particolare della (26). (26') d ~ 2 m ~ (~o -- R~) m , (26') che ~ un caso particolare della (26). 8. Sia ~' un'area piana semplicemente connessa, limitata da un con- torno s', appartenente ad un piano x', y', e supponiamo che si possa fare la rappresentazione conforme dell'area or' sopra un cerchio ~r ap- partenente al piano xy mediante le formole: x' .--p(x, y), y'--- q(x, y), ove p, q sono dei polinomi (armonici). ove p, q sono dei polinomi (armonici). Si voglia poi cercare la funzione u', regolare in ~' e che soddisfa alle equazioni : ~u = ~x, = ~x,2 + , ~'= o (,u ,9 ~u = ~x, = ~x,2 + , ~'= o (,u ,9 ove F' ~ una data funzione n-armonica (rispetto alle variabili x', y'). 116 TOMMASO BOGGIO. Converrgt fare la rappresentazione conforme di ~r' sul cerchio a, e si avr~t cosi: 117 1 s ) i 1 s ) i ove la costante b, ha lo stesso significato che nella (23). ove la costante b, ha lo stesso significato che nella (23). Dall'equazione precedente si ricava questa formola, analoga alla (24) : ove la costante b, ha lo stesso significato che nella (23). Dall'equazione precedente si ricava questa formola, analoga alla (24) : ?"+'-- (m--I)[h, o ~'(- I)'(m i 17 ~Jo ? -~fd? I m--t -- I~ _s_~_~ 3 t"9 s+i~ (s=o, i ..... "--0, e cosl conosciamo le funzioni armoniche %, ?m+,, "'" ? ..... 9 e cosl conosciamo le funzioni armoniche %, ?m+,, "'" ? ..... 9 1~ poi evidente che le equazioni (2L) , (2L) , ... (25,~) sussistono inalterate anche nel caso attuale di un campo sferico, perci6 da esse potremo ricavare le rimanenti funzioni armoniche ?o, ?z, ".', %-, ;e cosl otterremo la funzione u espressa mediante integrali semplici (an- zicM tripli). Se F 6 un polinomio, anche la u 6 un polinomio, e questa pro- prietk vale anche seil contorno , ~ un eUissoide qualunque. In particolare, se F-- I, si ha come ~ facile verificare: I R2)m U-- (2m + I)l (~2 -- , onde confrontando colla (9): fs 2~ l~)~ (27) GradS-- m(4m~ i, ({a= -- ) " (27) Converrgt fare la rappresentazione conforme di ~r' sul cerchio a, e si avr~t cosi: Converrgt fare la rappresentazione conforme di ~r' sul cerchio a, e si avr~t cosi: 1 • --" H 2F (in ~) ~, = o (su s) _r-v [ '3 P 'l % { '3 q'l -- \3x] --\OyJ' 1 • --" H 2F (in ~) ~, = o (su s) OVe : _r-v [ '3 P 'l % { '3 q'l -- \3x] --\OyJ' OVe : OVe : _r-v [ '3 P 'l % { '3 q'l -- \3x] --\OyJ' ed u, F indicano le funzioni u', F' espresse mediante le variabili x, y. ed u, F indicano le funzioni u', F' espresse mediante le variabili x, y. Ora la funzione F ~ notoriamente poliarmonica, rispetto alle varia- bili x y; e poich6 H" + un polinomio anche la funzione H'F sar~t ed u, F indicano le funzioni u', F' espresse mediante le variabili x, y. O l f i F i li i i ll i Ora la funzione F ~ notoriamente poliarmonica, rispetto alle varia- bili x, y; e poich6 H" + un polinomio, anche la funzione H'F sar~t poliarmonica, perci6 la funzione u si pu6 ottenere col metodo esposto nel ~ 5; se ne deduce poi una formola analoga alla (26). 9. Le proprietor stabilite nei w 5, 6, 7 si possono estendere facil- mente al caso di 3 o pifl variabili. Indichiamo perci6 con S la sfera di raggio R, c01 centro nell'ori- gine O delle coordinate, e con ~r il contorno di S; allora dovendo cer- care la funzione u, regolare in S, e che soddisfa alle equazioni: l a~.u = F (inS) du d'-~u u = d--~ = .... ~ n~_----i -- o (su ~), ore F(x, y, z~) ~ una data funzione n-armonica, converr~t esprimere la F colla formola seguente, analoga alla (15): ore F(x, y, z~) ~ una data funzione n-armonica, converr~t esprimere la F colla formola seguente, analoga alla (15): ll~t . F-- ,? f,, (?--x-f-y'+z'), ed u con una formola analoga alia (22); e si dedurrk cosi: g a,,,= Z, 4sr ,+ ~ : a u -- ~, 4 s 0 ,-§ d? oi • u-- ~ 4' (s -- I) S t~"-4 , , 3 ~972 -T cio~ : e poi e poi e cosl via; e si otterr~l poi l'equazione corrispondente della (23)che ~: SULLE FD-NZIONI DI GREEN D'ORDINE m. Costruzione delle funzioni di Green d'ordine m per campi speciali. Io. Consideriamo ancora alcuni campi speciali a 2 e a 3 dimen- sioni. Indichiamo, come dianzi, con ~ l'area circolare di centro 0 e di raggio R, e diciamo s il contorno di ~. Sia poi M un punto qualunque di ~ (polo di G,~), M' la sua im- magine rispetto ad s, e P un punto variabile qualsiasi dir Ponendo poi : Ponendo poi : p p = 0 M, ?~ = 0 P, 0 = M 0""~, (28) r = MP, r' -- M'P (28) TOMMASO BOGGIO. 118 risulta : (29) r 2 = i~' -71- p~ -- a p p, cos 0, R* R* r '~ ~1 +P~--2--o ~'c~ (29) r 2 = i~' -71- p~ -- a p p, cos 0, (29) percib se si considera la s : percib se si considera la s : (3 O) r, --- ~R r' , (3 O) si pub scrivere : p I 4 (31 ) r: =--~(R + ~2~: __ 2R2~tcos0), (31 ) ed eliminando 0 ira questa e la (29): 2 r 2 I 2 ,,, = + - pg(R" - 87). 2 r 2 I 2 ,,, = + - pg(R" - 87). Questa semplicissima identitA sar~t adoperata spesso in seguito. Essa intanto mostra che, essendo M fisso, comunque si muova il punto P in ,, la s r I a finita e diversa da zero nel cerchio r e che nei punti _P di s (cio~ per ?~ = R), si ha: ( ) r x --- r (su s). r x --- r (su s). Risulta poi dalla (3 o) chela funzione log r, ~ armonica nel cerchio r perci6 in base alla (33) si conclude che La funzione prellminare di GRime di I ~ ordine, cio~ F~ ~ precisamente log r,, e la ordinaria fun- zione di GREZ~r ~ quindi: r ,) . (34) G, = log 7~ r ,) . G, = log 7~ Si pub ancora notate chese il polo M cade nel centro 0 di ~ la (3 o) perde ogni significato, ma la (3 2) porge perb in tal caso: r~--R. I t. da cui segue subito il noto integrale che risolve il problema di DIRICnLE'r per il cer- chio. Costruzione delle funzioni di Green d'ordine m per campi speciali. Cerchiamo ora il valore nel punto P della funzione G,, il cui polo ~ il punto M; converfft percib costruire anzitutto la funzione pre- ) Si pub osservare the dalla (34) si ha, nei punti di s: dGx __ i dr r dG __ x (dr dr,) I d(r:--r z) dn r dn r x cln r dn dn "--~ 2r 2 dn ' quindi, daUa (32) : d G, R 9 __ ~2 dn ~ Rr 2 ' da cui segue subito il noto integrale che risolve il problema di DIRICnLE'r per il cer- chio. da cui segue subito il noto integrale che risolve il problema di DIRICnLE'r per il cer- chio. SULLE FUNZIONI DI GREEN D~ORDINE rn, 1I 9 liminare Fro, m-armonica in ~ (rispetto alle coordinate di P) e che ve- rifica nei punti P dis le equazioni (5,) del ~ 3, cioe: d i r,. d ~ (r .... log r) d n~ -- d n~ (i = o, i, = ..... m -- O. (Sx) Poniam 0 perci6 : m--I (35) r. = oZ,(r =- r:)'%_,, (35) ove ?,,,_i ~ una s da determinarsi, poliarmonica d'ordine m- i. E chiaro allora, a causa delia (32), chela funzione data dalla (35) effettivamente m-armonica in e. ove ?,,,_i ~ una s da determinarsi, poliarmonica d'ordine m- i. E chiaro allora, a causa delia (32), chela funzione data dalla (35) effettivamente m-armonica in e. Nei punti di s, cio~, in virtfi della (33), per r, -- r, si ha dalla (35) : (36) rm = ~, (36) rm = ~, d'altra parte per i= o la (5,) porge: r m = r =m-2 log r, sicch8 nei punti dis si ha: sicch8 nei punti dis si ha: ~,. = r 2m-2 log r, e poich~ la q~., deve essere m-armonica in., si pub porre, nell'area . : (37) % = r .... log r, ; e poich~ la q~., deve essere m-armonica in., si pub porre, nell'area . : (37) % = r .... log r, ; e poich~ la q~., deve essere m-armonica in., si pub porre, nell'area . : (37) % = r .... log r, ; (37) % = r .... log r, ; infatti, come si verifica subito, questa funzione 8 proprio m-armonica in % e, per la (33), sul contorno diventa eguale a r ..... log r. Costruzione delle funzioni di Green d'ordine m per campi speciali. infatti, come si verifica subito, questa funzione 8 proprio m-armonica in % e, per la (33), sul contorno diventa eguale a r ..... log r. Determiniamo ora la funzione qam_ ~ . Osserviamo perci6 che nei punti dis si ha dalla (35): drm c1% ( dr dr, (38) an -- du + 2.r-d-~n -- r,--d-~n )%_,, (38) cio~ ricordando le (37), (33): dr,,(2m2)r,m_,dr r=._3dr , (dr dr,) dn ~-~logr+ dn -q- zr tin dn dr,,(2m2)r,m_,dr r=._3dr , (dr dr,) dn ~-~logr+ dn -q- zr tin dn e confrontando colla (5,) per i = I : r2._3dr r=m_3dr ` (dr dr,) -- = 2 r %_, d n d n -d-n d ' da cui : equazione che deve esser soddisfatta nei punti di s; per6 siccome la funzione r .... 4 ~ poliarmonica d'ordine m- I (precisamente come la equazione che deve esser soddisfatta nei punti di s; per6 siccome la funzione r .... 4 ~ poliarmonica d'ordine m- I (precisamente come la TOMMASO BOGGIO. I20 %_,), si pub porre addirittura, anche nei punti di .: %_,), si pub porre addirittura, anche nei punti di .: 9) q~m-1 ~--- -- r2"-4 9 l q~m-1 ~--- -- r2"-4 9 l (39) Passiamo ora a determinare la funzione q~ .... . Dalla (35) si deduce, nei punti di s: arm d'%+2r dr a~r,~ %_, dn 2- dn ~ "d-n dnJ dn 2 V [ d r '~__ [ d r '~ cP r ~ r,'] (40) -1- [_\dn] _ dn~J %-' dn dn %-2, (40) ricordando poi la (5,) per i -- 2, sostituendo a %, %_, i valori gi~t ottenuti (37), (39) e riducendo, si ha: ricordando poi la (5,) per i -- 2, sostituendo a %, %_, i valori gi~t ottenuti (37), (39) e riducendo, si ha: (dr dr,'~ ~ __r,=_6[drV__ r,,~_6Zdr, V_2r~m_6[dr'~(dr,'~ 4_d-n dn ] %-~- %dn J -t" k dn J \d nJk d n ] ' onde : I rim--6 %_~ = -7 , perci6 possiamo assumere, anche nei punti di ~: perci6 possiamo assumere, anche nei punti di ~: %_~ --7 analogamente ~ facile ottenere: analogamente ~ facile ottenere: l 2m--8 %-3 -- T r I -- --v r ~'~-~-~i (i -- i, 2 ..... m -- I). ) Nel caso del cerchio e del semipiano, la funzione G 2 6 stata ottenuta, in altro modo, da[ Prof. LAURICELLA. Cfr. la sua Nora: Integrazione dell'equazione A 2 (A 2 u) =o in un campo diforma circolare [Atti Ace. Torino, vol. XXXI (I895-96), pp. lOiO-1OI8] e la sua Memoria gik dtata. Rend. C~rc. Ma:em. Palermo, t. XX (~9o5).- Stampato il 20 maggio x9o $. Costruzione delle funzioni di Green d'ordine m per campi speciali. %-i 2 l 2m--8 %-3 -- T r I -- --v r ~'~-~-~i %-i 2 l 2m--8 %-3 -- T r e in generale : e in generale : I -- --v r ~'~-~-~i (i -- i, 2 ..... m -- I). %-i 2 (i -- i, 2 ..... m -- I). Sostituendo nella (35) si ha: Sostituendo nella (35) si ha: m~ I 2 2xi r2m--2--21 F m -- r~m-2 log r~ -Jr- i-~-(r -- r~) del resto assai facile verificare che questa funzione soddisfa a tutte le condizioni richieste. Dopo cib la (7~) porge: del resto assai facile verificare che questa funzione soddisfa a tutte le condizioni richieste. Dopo cib la (7~) porge: r m-x I (4 I) G,~=r~m-~log'-~--~--~,-~t(r~--r:)'r:'~-~-~', (4 I) d~t nel punto P il valore delia G~ avente per polo il punto M, che d~t nel punto P il valore delia G~ avente per polo il punto M, Se M cade nel centro O, la r~ va sostituita con R, come si ~ vi- sto in fine del ~ io. SULLE FUNZIONI DI GREEN D~ORDINE TRY,. 12I 12. Dalla (3 2) risuka ancora che nel cerchio , si ha sempre: 12. Dalla (3 2) risuka ancora che nel cerchio , si ha sempre: 12. Dalla (3 2) risuka ancora che nel cerchio , si ha sempre: (4 2) r: > r ; (4 2) r: > r ; di qui si pub concludere chela funzione G ha un segno costante in ~;. di qui si pub concludere chela funzione G ha un segno costante in ~;. Infatti, per la G~, si ha dalla (41): I r2 r x ,), G 2 -- -~- (r~ -- r 2) -- log -7- che pu6 ancora scriversi: r l f"-;" .V2 G2--r ~ __~ Idv ' v tl I perci6 dalla (4 2) ne segue, in ogni punto di r perci6 dalla (4 2) ne segue, in ogni punto di r G2>o. Per la G 3 si trova: G3 = __ I (r 2 __ 4 r2 r~ + 3 r4) -- r4 log r, 4 r ' che pu6 esser scritta anche cosi: che pu6 esser scritta anche cosi: fI r* {'" (v2 -- I)2 d G m u~ dl onde daUa (4 2) si conclude, nel cerchio .: onde daUa (4 2) si conclude, nel cerchio .: G 3 <o. Costruzione delle funzioni di Green d'ordine m per campi speciali. In generale, la (4Q pu6 ancora mettersi sotto la forma: r I [~T(V2 __ i) m-~ (4I') Gm= (-- I)m r2"~-zJ, " v dr, (4I') perci6 daUa (4 2) si deduce che la fl~n~ione G ha un segno costante neI cercbio ,; e precisamente sem ~ pari si ha G m ~ o, e sem 6 dispari si ha invece G m < o. 13. Costruiamo ora la G,~ per l'area piana indefinita ,, limitata da una retta s. t~ chiaro che tale area pu6 ottenersi dall'area circolare prima con- x6 x6 TOMMASO BOGGIO. I22 siderata supponendo che il punto O si allontani indefinitamente; all0ra t~ e R tendono ad oo ed il loro rapporto ad I, perci6 la (3o) si ri- duce ad : siderata supponendo che il punto O si allontani indefinitamente; all0ra t~ e R tendono ad oo ed il loro rapporto ad I, perci6 la (3o) si ri- duce ad : r, = r', essendo r' la distanza del punto P dall'immagine (o simmetrico) M' del polo M rispetto aila retta s. Sostituendo quindi r' al posto di r, nelia (4 I) avremo, per l'area considerata, il valore in P della G m il cui polo ~ il punto M. Poiche anche in questo caso ~ verificata la (42), sussiste pure per la G il teorema del ~ precedente. 1~ facile determinate, per le due aree ora considerate, la funzione m-armonica in ~ e che sul contorno assume colle sue derivate normali successive dei primi m- I ordini, dei valori assegnati; tale funzione infatti risulta espressa dalla (8,) od (8',) nella quale la funzione G., data daUa (4i). 14. Risolviamo ora le questioni analoghe nel caso di 3 variabili. Sia S la sfera di centro O e raggio R; e ~ il contorno di S. Sia poi M un punto qua!unque di S, M' ha la sua immagine rispetto a % e Pun punto mobile qualsiasi di S. Costruzione delle funzioni di Green d'ordine m per campi speciali. Adoperando le notaaioni (28) 6 chiaro che sussisteranno tutte le formole (29)-(33) , mentre, invece delia (34), si avr~: I I 4) _ r r t I I _ r r t Ci6 posto, determiniamo nel punto P, la funzione G il cui polo il punto M; costruiremo perci6 dapprima la funzione preliminare F.~, che nei punti P di r soddisfa alle (5), cio6: d ~ r~ d i r =~-3 dr~ i -- du i (i---o, i ..... m-- I). (5) d ~ r~ d i r =~-3 dr~ i -- du i (i---o, i ..... m-- I). (5) A tale oggetto esprimeremo ancora r m mediante la (35); ne viene che la (36) sussiste ancora, e osservando che per i -- o si ha dalla (5) : Fm ~ r2m-3 , si deduce, nei punti di ~: ~m ~ r2m--3 Fm ~ r2m-3 , ~m ~ r2m--3 e poiche la funzione 9m deve essere m-armonica, si pu6 porre, nei punti diS: (45) ?m -- r:'~-' ; ?m -- r:'~-' ; SULLE FUNZIONI DI GREEN DORDINE m. 1213 infatti, questa funzione, come si vede subito ricorrendo alia (3o), ~ ap- punto ,;-armonica in S, e poi sul contorno vale r ..... 3. p p Per determinate la funzione %_, si osservi chela (38) vale an- cora, e confrontandoia colla (5) per i~--- I, si deduce, ricordando la (45) : .... 4 dr - 2,~ 4 dr, (dr dr,\ (2m -- 3)r ~ = (2m -- 3)r - 7-tin 27 2r \~ -~nJ~m_,, onde : 2 n, -- 3 rZm-5 , ~_~ _7_ 2 2 n, -- 3 rZm-5 , ~_~ _7_ 2 equazione che ~ verificata nei punti di ,~; e poich~ la funzioue q)m--, poliarmonica d'ordine m ~ I, si pub porte, nelia sfera S: equazione che ~ verificata nei punti di ,~; e poich~ la funzioue q)m--, poliarmonica d'ordine m ~ I, si pub porte, nelia sfera S: 2 m -- 3 2m-- 5 ?'-' = 2 r, . 2 m -- 3 2m-- 5 (46) ?'-' = 2 r, . (46) Determiniamo ora la funzione q~ .... ; basta osservare chela (4 o) ancor vera, poi sostituendo a 9,,, q~ .... i valori (45), (46), ricordando la (5) per i= 2, e riducendo si ha facilmente: (2m-- 3) (2m-- 5) r2m-7 ~m--2 --- 2 ! Costruzione delle funzioni di Green d'ordine m per campi speciali. 2, 2 ' equazione che deve esser soddisfatta sulla superficie ~; nei punti di S potremo assumere : equazione che deve esser soddisfatta sulla superficie ~; nei punti di S potremo assumere : (2 - 3) (2 m - s) 2+-v 9 q~m-~ -- 2 ! 2 ~ r~ ; in modo analogo ~ facile otteuere: in modo analogo ~ facile otteuere: (2 m -- 3) (2 m -- 5) (2 m -- 7) r~m-9, cpm_~ ---- 3!2 ~ rale: cpm e in generale: q~m-; --- (2m--3)(2m--5)"'" (2m--2i"3Vi!21 I)(2m--2i--IJr~m-3-2~-- - q~m-; --- (2m--3)(2m--5)"'" (2m--2i"3Vi!21 I)(2m--2i--IJr~m-3-2~-- - (i= I, 2 ..... m-- i). Sostituendo nella (35) si ottiene: q~m-; --- (2m--3)(2m--5)"'" (2m--2i"3Vi!21 I)(2m--2i--IJr~m-3-2~-- - (i= I, 2 ..... m-- i). (i= I, 2 ..... m-- i). (i= I, 2 ..... m-- i). Sostituendo nella (35) si ottiene: Fro= r .... 3+~_] (2m--3)(2m--5)'"(em--2i--I) r=~,-3-~;; x i i!2 / dopo ci6, per avere la funzione G basta sostituire nella (7) e si ha: (47) G,=r2,~-3 __rt~,~-3 + ; (2m--3)(2m--5)'"(2m--2~--I)i!2; r,'m-~'2;. dopo ci6, per avere la funzione G basta sostituire nella (7) e si ha: dopo ci6, per avere la funzione G basta sostituire nella (7) e si ha: (47) G,=r2,~-3 __rt~,~-3 + ; (2m--3)(2m--5)'"(2m--2~--I)i!2; r,'m-~'2;. (47) G,=r2,~-3 __rt~,~-3 + ; (2m--3)(2m--5)'"(2m--2~--I)i!2; r,'m-~'2;. Un altro metod0, completamente diverso, per costruire la funzione TOMMASO BOGGIO. I:24 G m nel caso delia sfera, ~ stato esposto dal Prof. MARCOLONGO nella sua N ota : Determinazjone della funzjone di GREEN di grado n, nel caso di una sfera ). In una Nora successiva ) egli ha dato l'espressione esplicita della G,~ ) sotto una forma diversa dalla (47), e ne ha dedotto alcune interessanti proprietY. G m nel caso delia sfera, ~ stato esposto dal Prof. MARCOLONGO nella sua N ota : Determinazjone della funzjone di GREEN di grado n, nel caso di una sfera ). In una Nora successiva ) egli ha dato l'espressione esplicita della G,~ ) sotto una forma diversa dalla (47), e ne ha dedotto alcune interessanti proprietY. 15. Anche nel caso della sfera ci6 si pu6 dedurre facilmente chela stante in S. *) Cfr. anche : ORLANDO, Sulla funzione n '~ di GREEN per la sfera [Giornale di Matemafiche di BATTAGLINI, vol. XLII (I9o4), pp. 292-296]. ) Rend. Acc. Lincei, vol. X, 2 ~ sere. I9OI, pp. I3I-I37. ) Sulla funzione di GREEN di grado n per la sfera [Rendiconfi del Circolo Ma- temafico di Palermo, t. XVI (I9o2), pp. 230-235]. In fine di questa Nora il Prof MAR- COLONGO cortesemente scrive: ~c Dopo la pubblicazione della mia Nota aU'Accademia c~ dei Lincei, il Dr. BOGGIO mi ha gentilmente informato di aver per suo conto riso- ~ Iuto, con metodo del tutto diverso, il problema della determinazione della funzione ~c G, per la sfera, il cerchio, il semi-piano, ecc. Tale metodo e alcune propriet~ delle G, sono esposti in una Memoria che egli ha compiuto da oltre un anno e the an- tc cora non ~ stampata >). I1 presente lavoro 6 appunto queUo di cui 6 cenno nelle linee ora citate; esso, e quello che gli far~ seguito, trovasi gi~ annunziato helle pag. 6 e x98 delle mie due prime note gia citate. ) Cfr. anche : ORLANDO, Sulla funzione n '~ di GREEN per la sfera [Giornale di Matemafiche di BATTAGLINI, vol. XLII (I9o4), pp. 292-296]. Costruzione delle funzioni di Green d'ordine m per campi speciali. sussiste la (42), cio~ r, > r; da funzione G m ha un segno co- Infatti dalla (47) si ha: (47) si ha: G 2 -- iversi: 2 -- S: I _ _ (r t _ ;.)2, 2 r x r==.~I I ["rv= - - I Jl .I 2 r v2 ~,v, G 2 < o , G 2 -- I _ _ (r t _ ;.)2, 2 r x the pub pure scriversi: onde, nel campo S: Per la funzione G 3 si ha: I G 3 ---g-~(r~- 6r;r'.+ 8r, r 3- 3r4), che pub mettersi sotto la forma : r I I. 3 t a7 G3 __ 2!2 2 r3j (V 2 -- I) d v t 7)2 ' sfera S: G~>o. r I I. 3 t a7 G3 __ 2!2 2 r3j (V 2 -- I) d v t 7)2 ' S perci6, nella sfera S: ) Rend. Acc. Lincei, vol. X, 2 ~ sere. I9OI, pp. I3I-I37. ) Sulla funzione di GREEN di grado n per la sfera [Rendiconfi del Circolo Ma- temafico di Palermo, t. XVI (I9o2), pp. 230-235]. In fine di questa Nora il Prof MAR- COLONGO cortesemente scrive: ~c Dopo la pubblicazione della mia Nota aU'Accademia c~ dei Lincei, il Dr. BOGGIO mi ha gentilmente informato di aver per suo conto riso- ~ Iuto, con metodo del tutto diverso, il problema della determinazione della funzione ~c G, per la sfera, il cerchio, il semi-piano, ecc. Tale metodo e alcune propriet~ delle G, sono esposti in una Memoria che egli ha compiuto da oltre un anno e the an- tc cora non ~ stampata >). I1 presente lavoro 6 appunto queUo di cui 6 cenno nelle linee ora citate; esso, e quello che gli far~ seguito, trovasi gi~ annunziato helle pag. 6 e x98 delle mie due prime note gia citate. SULLE FUNZIONI DI GREEN D~ORDINE m. I2~ In generate, la (47) pub porsi sotto la forma: In generate, la (47) pub porsi sotto la forma: r I (47') G,.= (-- I)m+~ I'3i5"" (2m--3)r"-3"~7(v'-- x)'-' i)! 2,~_, v ~ dr, quindi, ricordando la (4 2) si conclude chela funzione G,. ha un segno costante neIla sfera S; e precisamente sem ~ pari si ha G,. ~ o, e se m + dispari ~ invece G. ~ o. Costruzione delle funzioni di Green d'ordine m per campi speciali. Se il campo S, invece che una sfera, ~ lo spazio indefinito fimitato da un piano a, la funzione r, risuherh espressa dalla (43), ore r' rap- presenta la distanza del punto P dall'immagine (o simmetrico) M' di M rispetto al piano a; e la funzione G., sar~ ancora data dalla (47). Inohre sussister~i sempre II teorema, dimostrato dianzi, sul segno di G.. Per ciascuno di quesfi campi si ha poi, mediante la (8)od (8'), la funzione m-armonica in S e che sul contorno assume, colle sue derivate normali successive dei primi m- x ordini, dei vatori assegnati. i6. Cerchiamo ora la funzione di GREEN d'ordine m e di I~specie nel caso din variabili e di un campo sferico. i6. Cerchiamo ora la funzione di GREEN d'ordine m e di I~specie nel caso din variabili e di un campo sferico. Converr~t distinguere due casi: I ~ I1 numero n ~ dispari; oppure n ~ pari, ma per6 2m ~ n. 2 ~ I1 numero n ~ pari ed inoltre 2 m ~ n. 2 ~ I1 numero n ~ pari ed inoltre 2 m ~ n. Indicando con r~,,, la funzione preliminare di GRZEN d'ordine m e di I ~ specie avente per polo il punto M di S, essa dovr~t, nei punti del contorno soddisfare nel I ~ caso alle equazioni: q d ~ I" d i (r .... log r) "'"__ (i=o, t .... ra -- 0 d n ~ d n ~ q d ~ I" d i (r .... log r) "'"__ (i=o, t .... ra -- 0 d n ~ d n ~ kre: d i F d ~ r .... "'" (i=o, i .... m--O, dn ~ -- dn i (i=o, t .... ra -- 0 e nel 2 ~ caso alle akre: e nel 2 ~ caso alle akre: ore, al solito, r indica la distanza di M da un punto variabile qualun- que P di S. ore, al solito, r indica la distanza di M da un punto variabile qualun- que P di S. Dopo ci6 la funzione di GREEN Gin,,, d'ordine m e di I" specie a- vente per polo M sar~t data dalla formola: G,.,. = r .... -- r,.,., che 6 l'analoga della (7); ovvero daU'ahra : (7',) G,... -- r .... log r -- r,.,., che 6 la corrispondente della (7,). i"26 TOM. MASO BOG~IO. ) POINCAR~, Theorie du Potentiel newtonlen, S 73 (Paris, 1899 ). Costruzione delle funzioni di Green d'ordine m per campi speciali. Adoperando le stesse notazioni che nel ~ IO, si conclude chela funzione Gin," 4 in ogni caso data dalla formola seguente, che 4 una generalizzazione &lie (41'), (47'1: g r I r .... (v~ -- dr, (48) O,,,,,, = k,,,,,, v"-' (48) ove k.,. ~ una costante numerica, che ora determineremo. Poichs nel campo considerato ~ soddisfatta la (42), cio~ r x > r, si trae dalla (48) chela funzione O,~,. ha un segno costante nel campo considerato, e precisamente ha il segno della costante k m~n m n g facile trovare iI valore di questa costante. Infatti nel I ~ caso si ha dalta (48), eseguendo l'integrazione: m--t ' -- -....L.: -- I (48') cio~ : G=,.=<. oZ,(-O m--1 ' i 2 m -- n-- 2 i (r=i r~= ..... i _ r .... ) ; in questa formola il coefficiente di r .... ~: in questa formola il coefficiente di r .... ~: _l ( ) k .... ~;(--I1' m--I -i i 2m- n -- 2i ' onde, confrontando cola (7') si deduce: onde, confrontando cola (7') si deduce: I km, n 2 -- m--i o~i( - i)i(m --I) I i 2m--n--2i Nel 2 ~ caso, posto per brevitt 2p--2m- n, si ha invece della (48'1 la seguente: . . . . I z [{ r.~' ..... ' k r .... IX;-,.( - (m)2m __ n__ 2iL\_7/ --I] O=,= ,-,. t~o '" z)~ i "~ (-- I)P (m p I Ore cola scrittura ~-'~ si intende che la sommatoria va fatta escludendo il valore i--p; si pu6 ancora scrivere: Ore cola scrittura ~-'~ si intende che la sommatoria va fatta escludendo il valore i--p; si pu6 ancora scrivere: ,o - (T) 9 2m -- n -- 2i (r=irl ..... i r .... ) +(--0~(m/) --I) r=,,,_,,(logr _logr)l ; I2 7 SULLE FUNZIONI DI GREEN D~ORDINE ~. I2 7 in questa formola il coefficiente di r ..... log r vale: )' in questa formola il coefficiente di r ..... log r vale: )' I k~,.(-- I)P( m --p ~)' quindi; confrontando colla (71) otteniamo : quindi; confrontando colla (71) otteniamo : Si pu6 infine osservare che essendo la (32), al pari della (zg) , simme- trica rispetto a e e e~, cio~ ad M e P, ne viene che la stessa proprietY. compete atla funzione G .... Costruzione delle funzioni di Green d'ordine m per campi speciali. il che ~ in armonia cot teorema di recipro- cit~ relativo aUe funzioni di GREE~. 17. Dalla formola (48) ~ facile dedurre una propriet~ della fun- zione G,,,,., che ~ un'estensione di un'akra dovuta al POmCAa~ ), reIa- tiva alla ordinaria funzione di GREta. Consideriamo, per semplicitft, il caso di 3 variabili. Indichiamo con S ed S' due sfere di cui la seconda sia interna alla prima; sia poi M un punto fisso qualunque di S' e diciamo G la funzione di GREE~ d'ordine m e di I" specie, retafiva ad S, e avente per polo M, e G' quella relativa ad S' e avente pure per polo M. q p p p Sussiste altora la proprietk seguente : ]n ogni punto di S' si ha: O,. ~ G',., se m ~ dispari, G,. ~ G',., sem 8 pari. O,. ~ G',., se m ~ dispari, G,. ~ G',., sem 8 pari. O,. ~ G',., se m ~ dispari, G,. ~ G',., sem 8 pari. r I la dalla (47') : rL Infatti conservando le notazioni dei ~w precedenti e indicando con funzione analoga ad rx, ma relativa per6 alla sfera S', si ha ( ) r.L r r I onde : r I (49) G..- G' ( ~.~ .... ~.3.s ... ~-'2--3)r ..... ~ F( v~ =.- . (,~ - ~) !2 ~-' &: dr, dr, __ i) rn-~ V2 d v; ) POINCAR~, Theorie du Potentiel newtonlen, S 73 (Paris, 1899 ). TOMMA$O BOGGIO. I28 (50) r, > r',, (50) questa diseguaglianza si pub verificare facilmente con un calcolo diretto; I per6 si possono evitare calcoli osservando che -- ~ ta ordinaria fun- I zione preliminare di GRzz~r relativa ad S e avente per polo M, ed r-- 7- I l'analoga funzione relativa ad S' e avente pure per polo M; ma per questo caso il teorema in questione 6 veto, come ha stabilito il POINCAR~, cio6 ! < r~-~__, di qui segue la (5o), e daUa (49) si deduce poi sen- /'I I z'altro : G,. -- G' m ~ o, sem e dispari, G m ~ Grin ~ o, sem ~ pari, conforme al teorema. conforme al teorema. conforme al teorema. Studio deUe derivate successive delle funzioni di Green d'ordine m. I8. Mediante ie formole (4I), (47) ~ facile trovare dei limiti su- periori per le derivate successive delia funzione G m relativa ad un campo circolare o sferico. Stabiliremo anzitutto alcune diseguaglianze relative alle derivate suc- cessive della funzione G I . Consideriamo perci6 nuovamente un cerchio ~ di raggio R, ii cui centro O ~ l'origine delle coordinate; al solito, diremo s il contorno di ~. Usando le stesse notazioni che a ~ Io, la funzione G, ~ data dalla (34), cio~ : G, -- log r -- log r,. Siano poi (x, y) le coordinate del polo M, (4, ~) queue del punto mobile P, e cerchiamo le derivate successive di G rispetto ad x, y. Indichiamo perci6 con + l'angolo delia direzione P M coll'asse O x; ailora poich4 : r = = (x -- ~)~ -l- (Y -- ~)=, si ha: Indichiamo perci6 con + l'angolo delia direzione P M coll'asse O x; ailora poich4 : r= = (x ~)~ l (Y ~)= si ha: lora poich4 : r = = (x -- ~)~ -l- (Y -- ~)=, si ha: oich4 : r = = (x -- ~)~ -l- (Y -- ~)=, si ha: Or =cos~ =x--~ Or O---x r ' O--y -- sen hb -- y -- ~ Or =cos~ =x--~ Or O---x r ' O--y -- sen hb -- y -- ~ SULLE FUNZIONI DI GREEN D'ORDINE m. 12 9 se ne trae facilmente: se ne trae facilmente: 31ogr __ I COS + Ox r 82 log r __ I 8 ~ Ox: ----7 c~ 2+= logr c)y 2 0 log r __ I sen + Oy --7 & log r __ I Oxay -- - r --i- sen 2+, 82 log r __ I 8 ~ Ox: ----7 c~ 2+= logr c)y 2 & log r __ I Oxay -- - r --i- sen 2+, e in generale: e in generale: e in generale: a" log," -- i)! F-F - (- O"-'(n F cos,~+ (" = ', = .... ), a" log r I)"-' (n -- I) l Ox"-'3y -- (- r" senn+ a" log," -- i)! Rend. Circ. Matera. Palermo, t. XX (zgoi). --Stampato il ao maggio i9o 5. Studio deUe derivate successive delle funzioni di Green d'ordine m. F-F - (- O"-'(n F cos,~+ (sO a" log r I)"-' (n -- I) l Ox"-'3y -- (- r" senn+ (sO le altre derivate d'ordine n non hanno un valore diverso da quelle ora scritte; indicando quindi con D" una derivata parziale qualsiasi d'ordine n, rispetto alle variabili x, y, cio~ uno qualunque dei simboli 3" 3" 3" 3" 3x" ' 3x"-'3y' "'" 3xOy"-' ' 3y" ' vremo daUe (St): [D"logrl < (n-- I)l r" 3" 3" 3" 3" 3x" ' 3x"-'3y' "'" 3xOy"-' ' 3y" ' avremo daUe (St): [D"logrl < (n-- I)l r" Veniamo ora alle derivate di log r,. Ponendo -~- -- % si deduce dalla (32): ( ) (3 2') r~ --- r ~ --}- (I -- ~)(R 2 -- 72), r~ --- r ~ --}- (I -- ~)(R 2 -- 72), perci6 derivando e riducendo: perci6 derivando e riducendo: 3r, 3r, r' ox _~o~xm;, r c)y __~2y__~, 3r, 3r, r' ox _~o~xm;, r c)y __~2y__~, onde : a , 3 , 0~ R4 0), quindi, per la (31): ( onde : onde : a , 3 , 0~ R4 0), quindi, per la (31): ( 3r "~ ~ . l' Or,\ 2 o ~ ( 3r "~ ~ . l' Or,\ 2 o ~ ne segue che si pu6 trovare un angolo +, tale the: ne segue che si pu6 trovare un angolo +, tale the: 3r, 3r, 0~cos+, -- 0x ' ~senq; -'-3y Osserviamo ora, ci6 che del resto 6 facile verificare, che da ogni eguaglianza contenente x, y, r, d? se ne ottiene una nuova sostituendo Rend. Circ. Matera. Palermo, t. XX (zgoi). --Stampato il ao maggio i9o 5. I7 TOMMA$O BOGGtO. IJO al posto di queste quattro quantitA rispettivamente queste ahre: ~x, ~=y, -r,, +,. Quest'osservazione permette di dedurre subito dalle (51), senza eseguire nuovi caicoli, le seguenti eguaglianze : o~" log r, I)"-' (n -- I) a x" -- (- ! ~" cos n +, r7 0"logr x 1)._~(n-- I)! . ~x._ic3y__ (-- rx" sen n+~ da cui segue: o~" log r, I)"-' (n -- I) a x" -- (- ! ~" cos n +, r7 (n-- t, 2 .... 0"logr x 1)._~(n-- I)! . ~x._ic3y__ (-- rx" sen n+~ ID"logr,[ < (n-- I)! 0~" r]' od ancora, per la (42): [D. logrx I~(n-I)! . ?.n (n-- t, 2 .... ), da cui segue: da cui segue: ID"logr,[ < (n-- I)! Studio deUe derivate successive delle funzioni di Green d'ordine m. 0~" r]' 2): [D. logrx I~(n-I)! . ?.n od ancora, per la (42): ) [D. logrx I~(n-I)! . ?.n Ricordando l'espressione di G, deduciamo poi: Ricordando l'espressione di G, deduciamo poi: Ricordando l espressione di G, deduciamo poi: (52 ) [D"G,[<(1-I-o~") (n r .1)! (n=x, 2 .... ). (52 ) [D"G,[<(1-I-o~") (n r .1)! (52 ) (n=x, 2 .... ). I9. Riprendiamo ora la (4 I) che pub anche scriversi: n~ -'t ~ 21 ~.2m--2--2i y2m--2 o~,=4, ,r ~ + o~, le a~ essendo costanti numeriche conosciute. le a~ essendo costanti numeriche conosciute. La ~- che comparisce nel 2 ~ membro 6 un polinomio omogeneo di grado 2m -- 2 in x, y, ~, ~, R, come si vede subito ricorrendo alia (32'); chiamando P(x, y) tale polinomio, potremo ancora scrivere: O.=P+r .... G,; derivatorio D ~ + facile avere un risultato della operando col simbolo fos : fos : i (53) D ~ G~, = D~ P--} - ~.o ihiDJr~"-~D~-J G I , (53) le h i essendo costanti numeriche; ora DiP ~ un polinomio omogeneo di grado 2m--2--i in x,y, ~,'~, R, e poich~ x,y, ~, ~ non su- perano R potremo scrivere evidentemente : le h i essendo costanti numeriche; ora DiP ~ un polinomio omogeneo di grado 2m--2--i in x,y, ~,'~, R, e poich~ x,y, ~, ~ non su- perano R potremo scrivere evidentemente : aiR , se i Z 2m -- 2, lea' essendo humeri positivi, ed: lea' essendo humeri positivi, ed: DiP= o, se i~ 2m -- 2; si ha poi ancora : IDir~"-" I ~ b' r ='-'-j i , ~e jL2~ --~, SULLE FUNZIONI DI GREEN D~ORDINE m. 13I le b'i essendo pure numeri positivi, ed le b'i essendo pure numeri positivi, ed le b'i essendo pure numeri positivi, ed DJr .... = o, se j > 2m -- 2. DJr .... = o, se j > 2m -- 2. j Applicando la (52) abbiamo quindi: ] D i r .... D '-i G, ] < b;i i r ..... , ; ] D i r .... D '-i G, ] < b;i i r ..... , ; sostituendo nella (53) otteniamo un risultato della forma: sostituendo nella (53) otteniamo un risultato della forma: tD' 0~1 < a;R ....... '+ b;;~r .... -'<,~;'R ..... Studio deUe derivate successive delle funzioni di Green d'ordine m. ' (~_Z2m-~), b ftr ID:~-'G i<--, r d l i i i i i b ftr ID:~-'G i<--, r a'i' , b'" essendo altri numeri positivi. a'i' , b'" essendo altri numeri positivi. Se ne deduce con somma facilit~: Se ne deduce con somma facilit~: f]D' Go, I d ~ < c, R .... ' (i = i, 2 ..... ~ m -- ~), (54) (i = i, 2 ..... ~ m -- ~), c i essendo altri humeri positivi. Si ottiene poi per i = o una disugua- glianza analoga alla precedente ricorrendo alla (26'). c i essendo altri humeri positivi. Si ottiene poi per i = o una disugua- glianza analoga alla precedente ricorrendo alla (26'). t~ chiaro che i limiti superiori ora trovati dipendono esclusivamente dal cercbio e, e tendono a ~ero qualzdo il raggio decresce indefinitamente. Ci6 posto, sia F(x, y) una funzione data, continua colle sue deri- rate prime, e consideriamo la funzione u, regolare in ~, che soddisfa alte equazioni : o : l A U _= F (in ~) du d"-~u -- = .... d n ''-----r -- o (su s) ; U=dn essa ~ data, come abbiamo visto, dalla (%), cio~: = flo~(x, y, ~, ~)F(~, ~)d., k' u(x, Y) dalia quale si ha: k'Diu --JD~G,,.Fd* (i=~, 2 ..... 2m-0. k'Diu --JD~G,,.Fd* (i=~, 2 ..... 2m-0. Chiamando ~I, il massimo valore assoluto di F in r e ricordando the la funzione (~ I)" G,, ~ sempre posifiva, qualunque sia m, nel cer- chio ~ (~ 12), si ha: k'[u I <(-- i)"* f G.,d,, tiff 6'): ed applicando la (26'): t.l < co(R * -- P')~', t.l < co(R * -- P')~', ove c o indica una costante positiva. I~2 TOMMASO BOGGIO TOMMASO BOGGIO. I~2 Dalla (54) abbiamo poi: k'lD'u] <c,R'-i'l ' (i=i, 2 ..... m--i), k'lD'u] <c,R'-i'l ' (i=i, 2 ..... m--i) sicch~ indicando con ),~ una costante numerica positiva, possiamo scri- vere : ID'ul <),,R2"-"t ' Ci=o,, ..... 2m-x). (55) ID'ul <),,R2"-"t ' Ci=o,, ... ID'ul <),,R2"-"t ' Risulta evidente, da queste diseguaglianze, che le derivate parzjali, dei primi 2 m -- I ordini, della funzione u, sono finite ancbe nei punti del contorno, e tendono, in valor assoluto, a zero quando iI cercbio (z decresce i ndefinitamente. 20. Stabiliamo ora le proprietor analoghe nel caso di una sfera S. Studio deUe derivate successive delle funzioni di Green d'ordine m. Abbiamo gi~t visto chela funzione G avente per polo il punto M di S ~ data dalla (44), cio~: Cr -- I I r r~ Siano poi (x, y, K) le coordinate del polo M, (E., ~, ~) quelie del punto mobile P, e cerchiamo deUe limitazioni per le derivate successive di G,, rispetto ad x, y, ~:, e per questo incominciamo dalla funzione I. r Ponendo : Ponendo : X~x--~, Y--y--~, Z---Z~, r'---X'+ y'+ z ", avremo : avremo : onde la funzione ! soddisfa, rispetto alle variabili X, Y, Z, all'equazione r onde la funzione ! soddisfa, rispetto alle variabili X, Y, Z, all'equazione r di LA~LACR~, ed a omogenea di grado --I; ne segue che le sue derivate di un ordine qualunque n rispetto alle variabili x, y, Z, o, ci6 che ~ lo stesso, rispetto alle variabili X, I', Z saranno funzioni omogenee di X, Y, Z di grado --n- I; indicando, come gik dianzi, con D" una qua- lunque di queste derivate d'ordine n ~ facile vedere (del resto ~ ben noto) che si avr~.: D. fi. L __ P.(X, Y, Z) r r 2n'+'x ove P, ~ un polinomio armonico, omogeneo di grado n. Se poi si fa la trasformazione in coordinate polari: X=rsen0cos% Y--rsen0sen% Z=rcos0, si pub scrivere: Y % p. = r Y. % SULLE FUNZIONI DI GREEN D'ORDINE m, I~ essendo Y (% 0) una funzione sferica d'ordine n; quindi avremo: (56) D.!= i r r "+' Y" (% 0). (56) Cerchiamo ora un'espressione analoga per D "I. Dalla (32') 6 facile rt trarre procedendo come a ~ I8: Cerchiamo ora un'espressione analoga per D "I. Dalla (32') 6 facile rt trarre, procedendo come a ~ I8: trarre, procedendo come a ~ I8: + :e, onde si possono trovare due angoli c?,, 01 tall che: onde si possono trovare due angoli c?,, 01 tall che: Or~ 0r_~, __ Or, Ox = ~ sen 0 cos %, c~y -- ~ sen 0 sen ~I' ~ : ~ COS 0i. In virtfl di un'osservazione fatta nel ~ 18, si deduce aUora senz'altro dalla (56) : In virtfl di un'osservazione fatta nel ~ 18, si deduce aUora senz'altro dalla (56) : D" I ~" - 03. Studio deUe derivate successive delle funzioni di Green d'ordine m. I A~R~"-3-+ se iL2m--3, 12r2m--3_~_ ~- a rair2m--3--2tl < A, r2m_3_ i -- oZ~i i t II + se i> 2m-- 3, r j r j r j r2m-3-i-J ~ ~ -- r~m-3-~; x r~_(2m_3_i) rj_(2m_3_i) r j r j r j r2m-3-i-J ~ ~ -- r~m-3-~; x r~_(2m_3_i) rj_(2m_3_i) se poi i~2m--3 si ha: r um-3-i ~ a'"i Ram-3-1, con a'"i ~ O, sicch~ concludendo : t a" R 2'~-3-I ri r~m_3_~_i < i . se i ~ 2 m --- 3, r 2m-3-' se i~ 2m ~ 3; perci6 : D~ l ~-2 . I A~R~"-3-+ se iL2m--3, 12r2m--3_~_ ~- a rair2m--3--2tl < A, r2m_3_ i -- oZ~i i t II + se i> 2m-- 3, '/i '/' sono numeri positivi r j r j r j r2m-3-i-J ~ ~ -- r~m-3-~; x r~_(2m_3_i) rj_(2m_3_i) se poi i~2m--3 si ha: r um-3-i ~ a'"i Ram-3-1, con a'"i ~ O, se poi i~2m--3 si ha: r um-3-i ~ a'"i Ra se poi i~2m--3 si ha: r um-3-i ~ a'"i Ram-3-1, con a'"i ~ O, h l d d sicch~ concludendo : oncludendo : t a" R 2'~-3-I ri r~m_3_~_i < i . se i ~ 2 m --- 3, r 2m-3-' se i~ 2m ~ 3; t a" R 2'~-3-I ri r~m_3_~_i < i . r 2m-3-' perci6 : p D~ l ~-2 . I A~R~"-3-+ se iL2m--3, 12r2m--3_~_ ~- a rair2m--3--2*tl < A, r2m_3_ i -- oZ~i i t II + se i> 2m-- 3, ore ~'/i, z'/' sono numeri positivi. ore ~'/i, z'/' sono numeri positivi. Si riconosce poi facilmente, applicando la (57), e procedendo come a ~ 19, che: a 19, che: ID'(r .... G,) I < b, P'~-'-'. Si deduce quindi dalla (58): b' R ~'~-3-+ (i L 2 m 3), ID~Gml < ~ _ - b" b'" [D~m-~G"] ~ -'-7"' [D .... G,~] ~ -7 ' (i L 2 m 3), _ - le b essendO numeri positivi; se ne trae: le b essendO numeri positivi; se ne trae: fS iG 2m-i ID raids < cig (i= x, 2 ..... 2m-- x), ove c~ indica un akro numero positivo. ove c~ indica un akro numero positivo. Nel caso in cui i= o si ottiene la disuguaglianza analoga dalla (27). Nel caso in cui i= o si ottiene la disuguaglianza analoga dalla (27). Studio deUe derivate successive delle funzioni di Green d'ordine m. r t r~ +x Ricordando l'espressione di 0 avremo dunque: Ricordando l'espressione di 0 avremo dunque: D" i---r , < "r "h ~- i (57) ID"GI < D"+ + (57) ore b. + una costante numerica positiva ; questa diseguaglianza 6 l'ana- ioga delia (52). ore b. + una costante numerica positiva ; questa diseguaglianza 6 l'ana- ioga delia (52). 21. Riprendiamo ora la (47) che pub anche esser posta sotto la forma : 21. Riprendiamo ora la (47) che pub anche esser posta sotto la forma : (5 8) G,, -- ar ="-3 -1- ;alr2ir~ "-3-~; + br .... G,, (5 8) le a, b essendo costanti numeriche conosciute. le a, b essendo costanti numeriche conosciute. Considerando l'espressione entro le {.-. } 6 facile trarne: D ~ a r 2"-3 -Jr- ~o; a; r'; r~ "-~-2; < i r, le a'. essendo humeri positivi; ci6 posto consideriamo il termine: l le a'. essendo humeri positivi; ci6 posto consideriamo il termine: l rJ r~m--3--~--! ; se/L 2m--3- i (sar.~ anche iZ 2m- 3), l'esponente di r, positivo o nullo, e poich+, come risuka dalla (32'), si ha: o~r, < R ffS", o~r, < R ffS", ne segue : R 3 1 ne segue : ne segue : - -X tP R~m--3--1 r/r ="-3-~-j ~ a~ (iZ2m-- 3). TOM~tASO BOOGIO. 134 ove a"~ 8 un numero positivo ; se invece j ~ 2 m -- 3 -- i, l'esponente di r, ~ negativo, e poich~, come sappiamo, r ~ r, avremo: ove a"~ 8 un numero positivo ; se invece j ~ 2 m -- 3 -- i, l'esponente di r, ~ negativo, e poich~, come sappiamo, r ~ r, avremo: ove a"~ 8 un numero positivo ; se invece j ~ 2 m -- 3 -- i, l'esponente di r, ~ negativo, e poich~, come sappiamo, r ~ r, avremo: r j r j r j r2m-3-i-J ~ ~ -- r~m-3-~; x r~_(2m_3_i) rj_(2m_3_i) se poi i~2m--3 si ha: r um-3-i ~ a'"i Ram-3-1, con a'"i ~ O, sicch~ concludendo : t a" R 2'~-3-I ri r~m_3_~_i < i . se i ~ 2 m --- 3, r 2m-3-' se i~ 2m ~ 3; perci6 : D~ l ~-2 . Torino, gennajo x9o 5. Studio deUe derivate successive delle funzioni di Green d'ordine m. 1~ chiaro che tail Iimiti superiori dipendono solo dal campo S, e ten- dono a <ero quando il raggio decresce indefinitamente. 1~ chiaro che tail Iimiti superiori dipendono solo dal campo S, e ten- dono a <ero quando il raggio decresce indefinitamente. Se ora si indica con F(x, y, <) una funzione data, continua colle sue derivate prime, e si considera la funzione u, regolare nella sfera S, e che soddisfa alle equazioni: • u -- F (in S), du dm-'u u = d--~ = .... d n +-----i (su ,), essa verificher~t le disuguaglianze seguenti, analoghe alle (55): essa verificher~t le disuguaglianze seguenti, analoghe alle (55): ), R2"-iO (59) ID'ul < , ), R2"-iO (i=o, I .... 2m-- i), (59) ID'ul < , , (59) SULLE FUNZIONI DI GREEN D~ORDINE m. I55 ove 9 ~ il massimo valor assoluto di F in S e le ),~ sono costanti nu- meriche positive. Queste disuguaglianze mostrano chiaramente che le derivate parz~iali dei primi 2 m -- I ordini delia fun#one u sono finite anche nei punti deI contorno e tendono, in valor assoluto, a zero quando la sfera S decresce indefinitamente. Questo teorema, e l'analogo enunciato in fine del ~ 19, sono esten- sioni di teoremi noti, relativi alla soluzione u dell'equazione ~2 u -- F, che si annuUa sul contorno del campo considerato. To~tAso BoGalO.
https://openalex.org/W4306874328	https://www.nature.com/articles/s41467-022-33659-1.pdf	English	null	Rapid antibiotic susceptibility testing and species identification for mixed samples	Nature communications	2,022	cc-by	8,645	Rapid antibiotic susceptibility testing and species identiﬁcation for mixed samples Vinodh Kandavalli1,2, Praneeth Karempudi1,2, Jimmy Larsson1 & Johan Elf 1 Vinodh Kandavalli1,2, Praneeth Karempudi1,2, Jimmy Larsson1 & Johan Elf 1 Received: 10 September 2021 Accepted: 28 September 2022 Check for updates Antimicrobial resistance is an increasing problem on a global scale. Rapid antibiotic susceptibility testing (AST) is urgently needed in the clinic to enable personalized prescriptions in high-resistance environments and to limit the use of broad-spectrum drugs. Current rapid phenotypic AST methods do not include species identiﬁcation (ID), leaving time-consuming plating or culturing as the only available option when ID is needed to make the sensitivity call. Here we describe a method to perform phenotypic AST at the single-cell level in a microﬂuidic chip that allows subsequent genotyping by in situ FISH. By stra- tifying the phenotypic AST response on the species of individual cells, it is possible to determine the susceptibility proﬁle for each species in a mixed sample in 2 h. In this proof-of-principle study, we demonstrate the operation with four antibiotics and mixed samples with combinations of seven species. The rapid increase in antibiotic resistance is a serious threat to human health; access to effective antibiotics is a cornerstone of modern medicine and a prerequisite for e.g. cancer treatment and surgery. Different investigations1,2 make different estimations of how grave the situation is but there is a consensus view that action needs to be taken or the costs both in terms of human suffering and global economic impact will be staggering3. Experts also agree that the problem is at least partly due to indiscriminate use and misuse of a wide range of antibiotics4. To overcome this problem, personalized and rapid anti- biotic susceptibility tests (ASTs) are needed, ideally at the point of care5. Without these tools, physicians are left with no other option than to prescribe broad-spectrum antibiotics in many cases since it can take several days to identify the pathogen(s) and the resistance proﬁle. not the main culprit, but rather the bulk usage of antibiotics for more benign conditions11. The exhaustion of effective antibiotics is also driven by the strategy to change ﬁrst-line antibiotics when the local resistance prevalence has reached approximately 10–20%. If fast, reli- able ASTs were accessible, high-resistance antibiotics could be used for 80–90% of the infections that are still susceptible. Article https://doi.org/10.1038/s41467-022-33659-1 1Dept. Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. 2These authors contributed equally: Vinodh Kandavalli, Praneeth Karempudi. e-mail: johan.elf@icm.uu.se 1Dept. Cell and Molecular Biology, Uppsala University, Uppsala, Sweden. 2These authors contributed equally: Vinodh Kandavalli, Praneeth Karempudi. e mail johan elf@icm uu se Article prevents the bacteria from escaping the trap while still allowing media and probes to ﬂow around the cells. To simulate a mixed infection situation, bacterial overnight cultures of several different species were diluted in a Mueller Hinton (MH) broth, pooled, and directly loaded into the microﬂuidic chip. In a typical experiment, loading one or a few cells in each trap takes 1 min at~105 CFU/ml. We supplied growth media with antibiotics to the traps in one of the two rows and plain growth media in the other. 2–6 h for positive blood cultures that have been growing >6 h from sampling the patient. For other samples, e.g., urine, the time-to-answer can be reduced to 30 min for gram-negative species by loading the sample directly into a microchip and measuring the growth rate with and without antibiotics15. A limitation of all rapid phenotypic AST methods is that they only work if the species of the bacteria are known. Detailed species ID is not needed for infections with a narrow spectrum of pathogens16, but for sepsis and other more complex infections, it is essential. MALDI-TOF mass spec is currently the golden standard for species determination17. MALDI-TOF does however still require pre-culture of single bacterial species and may not work well for mixed infections commonly seen in sepsis, wounds, catheter-associated UTIs18, and community-acquired pneumonia, among others19. The challenge remains to make rapid phenotypic AST with species ID. The phenotypic response to the antibiotic was determined in ≈60 min by capturing 100X phase-contrast images of each cell every 2 min and calculating the growth rates of individual cells in 10 min sliding windows. The phenotypic response can be pushed to shorter times depending on which antibiotics are used and at which concentration15. To identify the species of each bacterial cell, we per- formed ﬂuorescence in situ hybridization (FISH) using species-speciﬁc, ﬂuorescent ssDNA probes. These probes (Supplementary Data 1) bind to the abundant 16s/23s ribosomal RNA sequences and have previously been successfully used for species identiﬁcation in positive blood cultures21. The species classiﬁcation method for FISH signals is described in SI method 3. To address this issue, we use a microﬂuidic chip to rapidly capture individual bacteria from a sample and optically monitor their growth with and without antibiotics. Next, we identify the bacterial species by ﬂuorescence in situ hybridization (FISH) with species-speciﬁc ssDNA probes targeting the 16s/23s rRNA sequence. Phenotypic AST followed by Genotyping by FISH Phenotypic AST followed by Genotyping by FISH The culture chip that we have developed for this assay is capable of rapid capture of bacteria directly from a liquid sample and allows for optical monitoring of bacterial growth with and without antibiotics in real time. The same chip design has previously been used to capture bacteria from blood cultures despite an overwhelming excess of blood cells20. The chip features two rows of 3000 cell traps each. Each trap measures 1.25 × 1.25 × 50 μm15 and has a constriction at the end which A ground-truth training dataset (82 images) of mixed cells grow- ing in mother-machine devices was created with human supervision using the LabelsToROIs tool26 and custom scripts (SI method 1). We use an Omnipose model trained on this data for cell segmentation. Training and performance of Omnipose and U-net are also described in SI method 3. The run-time performance of reconstruction steps from Omnipose network outputs was improved two-fold (SI Fig. 1b). 0 min 60 mins 90 mins 120 mins Mixed species b Phenotyping c Genotyping d Analysis Treated Reference Species 2 Time Species 1 Time Time Species 3 a Microfluidics 130 mins e AST profiling Species 1 Species 2 Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Time (min) 10 20 30 40 50 Reference Treatment Reference Treatment Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Time (min) 10 20 30 40 50 Reference Treatment Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Time (min) 10 20 30 40 50 Species 3 Fig. 1 \| Schematic representation of the AST workﬂow with timeline. a A cartoon of the microﬂuidics setup with the mixed species loaded on the chip. b Time-lapse phase-contrast images of the cells in the traps when grown in media with (top) and without (bottom) antibiotics. c Fluorescence images of the bacteria with ssDNA probes targeting the ribosomal RNA of speciﬁc bacteria for species identiﬁcation d Analysis of time-lapse stacks and species ID using deep learning for segmenting and tracking cells. e Detection of AST proﬁles for individual pathogens at a given antibiotic concentration. Part of Fig. 1a created using www.biorender.com. Nature Communications\| (2022) 13:6215 2 Treated probes targeting the ribosomal RNA of speciﬁc bacteria for species identiﬁcation. d Analysis of time-lapse stacks and species ID using deep learning for segmenting and tracking cells. Analysis using Deep learning models Performing growth-rates analysis on multi-species samples requires a general method for detecting and tracking cells that come in different shapes and sizes. In Delta 2.022, a U-net23 was used to predict cell-vs- background maps of phase-contrast images and showed excellent performance for E. coli cells, both in mother-machine-like devices and on agarose pads. More general algorithms for cell segmentation, such as Cellpose24 and its successor Omnipose25 construct cell identities from intermediate ﬁelds (Fig. 2a) learned by a convolutional neural network, similar to a U-net. Omnipose was shown to perform seg- mentation of bacterial cells independently of their morphology. Approaches that calculate cell identities (Cellpose, Omnipose) assign different unique labels to cells that share a boundary while cell-vs- background approaches (U-net) can not be used to separate cells that share a boundary. The former approaches require cell labels as training data while the latter needs only binary masks. In this proof of principle application, we perform the ASTs for seven common pathogens (Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Proteus mirabilis, and Acinetobacter bau- mannii, as examples of gram-negative strains, and Enterococcus fae- calis and Staphylococcus aureus as examples of gram-positives). We test four different antibiotics from different classes: Vancomycin (Van) [Glycopeptide], Ciproﬂoxacin (CIP) [Fluoroquinolones], Gentamicin (Gen) [an aminoglycoside], and Nitrofurantoin (NIT) [other agents]. Finally, we show how single-round, multi-color labeling enables the identiﬁcation of up to ten species simultaneously. Article Once we have the species ID and AST response for each bacterium in the microﬂuidic chip, we stratify the AST response based on species, which implies that we can have samples with mixed species. A schematic overview of the approach is presented in Fig. 1. Rapid antibiotic susceptibility testing and species identiﬁcation for mixed samples The obvious need and beneﬁts of rapid AST, both for saving lives and guiding prescriptions, have resulted in the development of several new methods over the last decade. These methods are described in several recent reviews, e.g.,4,12, and we will not repeat all the pros and cons of the different methods here. Brieﬂy, these methods can be divided into two categories, genotypic and phenotypic. Genotypic methods identify speciﬁc genetic markers that are associated with antibiotic resistance. Although these methods can rapidly detect the presence of speciﬁc resistance genes, they depend on our knowledge of the resistance mechanisms which is far from complete13, in parti- cular considering the rapid emergence of new resistance mechanisms. Furthermore, the absence of resistance genes does not predict sus- ceptibility to antibiotics14, i.e., you may learn what not to use but not what will work. In phenotypic methods, the bacteria are exposed to an antibiotic and their phenotypic response, e.g., lysis or growth rate reduction, is monitored. Phenotypic methods work irrespective of the mechanism of resistance. If the phenotypic response is there, the bacterium is susceptible. The various rapid phenotypic ASTs that have reached the market can deliver an answer (susceptible or resistant) in The limitations of conventional phenotypic ASTs (disk diffusion agar dilution or broth microdilution) are that they require bacterial growth for extended periods in the presence and absence of anti- biotics to see an effect. However, for certain types of bacterial infec- tions, even a delay of 6 h before treatment is initiated can have severe consequences6. One such example is sepsis, where the risk of death has been estimated to increase by 7.6% for every hour that effective treatment is not given7. Further, it is shown that in the absence of fast AST, more than 25% of septic patients were treated by clinicians with inappropriate antibiotics, which is strongly associated with mortality8–10. Thus, rapid and accurate ASTs are needed to save lives. But considering the increases in AMR, life-threatening conditions are Nature Communications\| (2022) 13:6215 1 https://doi.org/10.1038/s41467-022-33659-1 Article Species-wise AST response With the species ID and AST response for each cell in the microﬂuidic chip, the species-speciﬁc AST response could be determined in the mixed samples. Here, we ﬁrst demonstrate the capability of the method to characterize a mixed sample of four different species, although clinical patient samples are more likely to contain only one or two30–32. The AST responses are shown in Fig. 3a–d. In each experiment, we obtained growth-response curves for three gram-negative strains (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa) and one gram-positive strain (Enterococcus faecalis). The experiments were performed with four different antibiotics: Vancomycin (Van) [Glycopeptide], Ciproﬂoxacin (CIP) [Fluoroquinolones], Gentamicin (Gen) [an aminoglycoside], and Nitrofurantoin (NIT) [other agents]. We present the results as response plots from individual experiments to simulate the clinical-sample situation. For comparison, we also display the average responses that would have been the result of the growth-rate measurements without species information. Successful AST proﬁling was achieved with samples containing as few as 100 bacteria/species. We used bacteria without speciﬁc resistance genes, but since some species have a natural resistance to speciﬁc antibiotics, the AST response varied with the species. For example, Pseudomonas species are naturally resistant to Ciproﬂoxacin (Fig. 3a) and Previous approaches to successful E. coli tracking have con- structed cell lineages using scoring mechanisms based on overlapping regions27 or mother-daughter binary-mask predictions using the full features from the images22. The former approach is very sensitive to tuning overlap parameters while the latter is computationally expen- sive when a lot of small cells are present in the data. Inspired by recent developments in using Siamese networks28 and graph formulations29 for general object-tracking, we have now developed an approach that performs cell tracking using a network that compares cell properties between two frames and predicts the presence and type of connec- tions, e.g., cell growth or division event (Fig. 2d). Training data for this network was obtained using a simulator (SI method 4) that simulates cells of various kinds and growth rates growing in mother-machine channels. The training of the tracking network is described in the Supplementary information (SI method 4). In Fig. 2e, we show the confusion matrix of the model used for tracking experimental data evaluated on 100 randomly generated time-series stacks. Before con- structing the tracks, the links that have abrupt increases in areas were removed as they correspond to merge events caused by segmentation errors (SI method 4). Phenotypic AST followed by Genotyping by FISH e Detection of AST proﬁles for individual pathogens at a given antibiotic concentration. Part of Fig. 1a created using www.biorender.com. Fig. 1 \| Schematic representation of the AST workﬂow with timeline. a A cartoon of the microﬂuidics setup with the mixed species loaded on the chip. b Time-lapse phase-contrast images of the cells in the traps when grown in media with (top) and without (bottom) antibiotics. c Fluorescence images of the bacteria with ssDNA Nature Communications\| (2022) 13:6215 2 2 Article https://doi.org/10.1038/s41467-022-33659-1 Segmentation workflow Segmentation metrics Segmentation comparisions Input Outputs + Conv-ReLU-Norm Skip connections Labels Reconstruction + + + flow-Y Boundary flow-X Distance Field Reconstruction Neural-net IoU matching threshold Jaccard Index U-net Omnipose Phase Ground Truth U-net Omnipose Cell-tracker network Tracker metrics Tracking of a single stack T T+1 Node features (T) Node features (T+1) No link Cell division Cell Movement Cell simulator Randomize Cell shapes, Growth rates, Frames Construct Dataset of Graphs Train/Test Edge prediction Netowrk Predicted labels No-link Movement Division True labels Confusion Matrix Time a b c d e f ig. 2 \| Analysis. a Omnipose network showing network structure, inputs, and utputs to the neural network and reconstruction of output to generate cell masks. Quantiﬁed performance of cell segmentation in mother-machine devices. Aver- ge precision vs IOU threshold plot for mixed-species dataset. The IOU threshold eﬁnes valid matching between predicted mask and ground-truth masks, 0.5 dicating half the pixels were correctly matched and 1 indicating pixel perfect match for every cell. Average precision (TP / (TP + FP + FN)) is calculated from the valid matches (TP), no valid matches (FP), and the ground-truth masks that have no valid match (FN). c Few examples of phase-contrast images, ground-truths and network predictions of U-net and Omnipose. d Overview of tracker network. e Confusion matrix of the edge predictions. f Tracks of cells in one channel of the mother-machine device. Phenotypic AST followed by Genotyping by FISH Segmentation workflow Input Outputs + Conv-ReLU-Norm Skip connections Labels Reconstruction + + + flow-Y Boundary flow-X Distance Field Reconstruction Neural-net a d Segmentation metrics IoU matching threshold Jaccard Index U-net Omnipose b Segmentation comparisions Phase Ground Truth U-net Omnipose Tracking of a single stack c f b c a Tracker metrics Predicted labels No-link Movement Division True labels Confusion Matrix e d d e Cell-tracker network T T+1 Node features (T) Node features (T+1) No link Cell division Cell Movement Cell simulator Randomize Cell shapes, Growth rates, Frames Construct Dataset of Graphs Train/Test Edge prediction Netowrk d Cell-tracker network Tracking of a single stack Time Time Fig. 2 \| Analysis. a Omnipose network showing network structure, inputs, and outputs to the neural network and reconstruction of output to generate cell masks. b Quantiﬁed performance of cell segmentation in mother-machine devices. Aver- age precision vs IOU threshold plot for mixed-species dataset. The IOU threshold deﬁnes valid matching between predicted mask and ground-truth masks, 0.5 indicating half the pixels were correctly matched and 1 indicating pixel perfect match for every cell. Average precision (TP / (TP + FP + FN)) is calculated from the valid matches (TP), no valid matches (FP), and the ground-truth masks that have no valid match (FN). c Few examples of phase-contrast images, ground-truths and network predictions of U-net and Omnipose. d Overview of tracker network. e Confusion matrix of the edge predictions. f Tracks of cells in one channel of the mother-machine device. machine trap tracked through time with growth/movement links (red) and division links (blue). Species assignment to tracks based on the species ID identiﬁed in the last frame was performed after cell-tracking (SI method 5). Omnipose showed superior performance (Fig. 2b) on mixed-species data compared to a U-net trained to predict binary masks. We showed that the omnipose performed equally well on mixed-species data as on data from pure E. coli cultures (SI Fig. 1b, e); the performance of U-net, on the other hand, depended on the species. Figure 2c shows phase contrast images, their corresponding ground truths, and cells detected using Omnipose and U-net approaches, respectively. https://doi.org/10.1038/s41467-022-33659-1 https://doi.org/10.1038/s41467-022-33659-1 K.pneumoniae (S)Treatment E.coli (S)Treatment P.aeruginosa (R) Treatment E.feacalis (S)Treatment No Species Id Reference No Species Id Treatment K.pneumoniae (S)Treatment E.coli (S)Treatment P.aeruginosa (S) Treatment E.feacalis (R)Treatment No Species Id Reference No Species Id Treatment K.pneumoniae (S)Treatment E.coli (S)Treatment P.aeruginosa (R) Treatment E.feacalis (R)Treatment No Species Id Reference No Species Id Treatment 10 20 30 40 50 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Ciprofloxacin 1μg/ml Time (min) 10 20 30 40 50 Time (min) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Gentamicin 2μg/ml K.pneumoniae (R)Treatment E.coli (R)Treatment P.aeruginosa (R) Treatment E.feacalis (S)Treatment No Species Id Reference No Species Id Treatment Nitrofurantoin 32μg/ml Vancomycin 4μg/ml a b d c Growth rate normalized Growth rate normalized Growth rate normalized Growth rate normalized Fig. 3 \| Species stratiﬁed responses to antibiotic treatments. a–d AST proﬁles with normalized growth rates for the four antibiotics used. The species stratiﬁed responses (mean and SEM), as well as the pooled response (without species stra- tiﬁcation), are shown for each antibiotic. In all AST proﬁle plots, S and R represent the Susceptible and Resistance, respectively. The experiment is performed once per antibiotic, although several non-reported experiments were performed for calibration. K.pneumoniae (S)Treatment E.coli (S)Treatment P.aeruginosa (S) Treatment E.feacalis (R)Treatment No Species Id Reference No Species Id Treatment 10 20 30 40 50 Time (min) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Gentamicin 2μg/ml b Growth rate normalized K.pneumoniae (S)Treatment E.coli (S)Treatment P.aeruginosa (R) Treatment E.feacalis (R)Treatment No Species Id Reference No Species Id Treatment 10 20 30 40 50 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Ciprofloxacin 1μg/ml Time (min) a Growth rate normalized Growth rate normalized 10 20 30 40 50 Time (min) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 K.pneumoniae (R)Treatment E.coli (R)Treatment P.aeruginosa (R) Treatment E.feacalis (S)Treatment No Species Id Reference No Species Id Treatment Vancomycin 4μg/ml d Growth rate normalized K.pneumoniae (S)Treatment E.coli (S)Treatment P.aeruginosa (R) Treatment E.feacalis (S)Treatment No Species Id Reference No Species Id Treatment 10 20 30 40 50 Time (min) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 Nitrofurantoin 32μg/ml c Growth rate normalized 10 20 30 40 50 Time (min) 1.0 0.8 0.6 0.4 0.2 0.0 Growth rate normalized Fig. https://doi.org/10.1038/s41467-022-33659-1 3 \| Species stratiﬁed responses to antibiotic treatments. a–d AST proﬁles with normalized growth rates for the four antibiotics used. The species stratiﬁed responses (mean and SEM), as well as the pooled response (without species stra- tiﬁcation), are shown for each antibiotic. In all AST proﬁle plots, S and R represent the Susceptible and Resistance, respectively. The experiment is performed once per antibiotic, although several non-reported experiments were performed for calibration. antibiotics (Fig. 5). For example, when Escherichia coli and Enter- ococcus faecalis were treated with Vancomycin (4 μg/ml), the two species showed clear, distinguishable growth-rate proﬁles corre- sponding to resistance and susceptibility, respectively (Fig. 5d). Repeats for these experiments are shown in Fig. 5e–h. Nitrofurantoin (Fig. 3b), and their growth remained unaffected in the presence of 1 μg/ml Ciproﬂoxacin or 32 μg/ml Nitrofurantoin. The growth rate of the other bacterial species dropped more than 10% in 30 min in the presence of these drugs. Importantly, we see from the average, non-species-stratiﬁed data that without access to species ID, we would not have been able to detect the resistant Pseudomonas in the mixed population. Similarly, as expected, all species but E. faecalis were found to be susceptible to Gentamicin (2 μg/ml) (Fig. 3c) and resistant to Vancomycin (4 μg/ml) (Fig. 3d). Nitrofurantoin (Fig. 3b), and their growth remained unaffected in the presence of 1 μg/ml Ciproﬂoxacin or 32 μg/ml Nitrofurantoin. The growth rate of the other bacterial species dropped more than 10% in 30 min in the presence of these drugs. Importantly, we see from the average, non-species-stratiﬁed data that without access to species ID, we would not have been able to detect the resistant Pseudomonas in the mixed population. Similarly, as expected, all species but E. faecalis were found to be susceptible to Gentamicin (2 μg/ml) (Fig. 3c) and resistant to Vancomycin (4 μg/ml) (Fig. 3d). Discussion In conclusion, we have demonstrated that it is possible to make rapid AST for mixed-species samples by performing sequential single-cell phenotypic susceptibility testing and ﬂuorescence in situ hybridiza- tion in a microﬂuidic chip. Importantly, ID determination is also useful for non-mixed samples when it is essential to know which MIC break- points to use for making the SIR (susceptible-intermediate-resistant) call. Although species determination for non-mixed samples is possi- ble by MALDI-TOF, it requires many more bacterial cells than direct single-cell imaging. Species-wise AST response Figure 2f (left) shows a single segmented mother- Nature Communications\| (2022) 13:6215 3 Article Scaling up FISH probing to 10 species Scaling up FISH probing to 10 species It is estimated that >90% of clinical sepsis samples feature a subset of the 10 most frequent bacterial pathogens33. To increase the number of species that we can identify, we performed combinatorial FISH using a species-speciﬁc adaptor that can bind two different ﬂuorescent oligo probes. With probes of four different colors, this set-up can identify up to 10 species (Fig. 4a). We demonstrated the combinatorial FISH approach by identifying seven species loaded in the culture chip (Fig. 4b). Species-wise combination of signals using all the adapters and probes are shown in SI Fig. 7. Species classiﬁcation was done using a random-forest classiﬁer on the 4-d signal obtained from stacking images of four ﬂuorescence channels (SI method 6). To demonstrate species-wise AST proﬁles using combinatorial FISH, we performed experiments with combinations of two species treated with different Since antibiotic susceptibility breakpoints of closely related spe- cies are often the same, it can sometimes be enough to distinguish the bacterial family or genera. For example, an Enterobacteriales-speciﬁc color ID-code to cover Escherichia, Klebsiella, and Salmonella, which have very similar resistance breakpoints34. Different species-speciﬁc probes would in this case map the same color ID code, such that the ten codes can be used more efﬁciently. Similarly, we expect that it would be possible to have a common ID code for contaminant species, Nature Communications\| (2022) 13:6215 4 Article https://doi.org/10.1038/s41467-022-33659-1 b a Pseudomonas aeruginosa Escherichia coli Enterococcus faecalis Ribosomal RNA Target sequence with multiple adapters Detection probes for the adapters Klebsiella pneumoniae Proteus mirabilis Acinetobacter baumannii Staphylococcus aureus Escherichia coli Klebsiella pneumonia Pseudomonas aeruginosa Enterococcus faecalis Acinetobacter baumannii Proteus mirabilis Staphyloccus aureus Phc Alexa 488 Cy3 Cy5 Texas Red Fig. 4 \| Combinatorial FISH. a Overview of the combinatorial FISH probing for the multi species identiﬁcation. A cartoon illustrating the different bacterial species with their ribosomal RNA (left). Illustration of the speciﬁc sequences with the multiple adapters targeting the ribosomal RNA of individual bacteria and its hybridization to the target rRNA (middle). Detection probes with different ﬂuor- ophores. Hybridization of detection probes to the adapter sequences along with unique sequences that are targeted to the species speciﬁc rRNA (Right). b Example mages (Scale bar 20 µm) of mixed species loaded in the microﬂuidic chip and probed using combinatorial FISH for species identiﬁcation. Scaling up FISH probing to 10 species After the hybridization step, cells were imaged in different channels (PhC, Alexa 488, Cy3, Cy5, and Texas Red). The bacterial species are marked in white (Escherichia coli), magenta (Kleb- siella pneumoniae), cyan (Pseudomonas aeruginosa), brown (Enterococcus faecalis), yellow (Acinetobacter baumannii), navy blue (Proteus mirabilis) and red (Staphy- lococcus aureus). The experiment with all seven species mixed was performed a single time. However, similar experiments with all adaptors and probes mixed are reported in Fig. 5. a Target sequence with multiple adapters Detection probes for the adapters Target sequence with multiple adapters Alexa 488 Ribosomal RNA Escherichia coli Enterococcus faecalis Cy5 Pseudomonas aeruginosa Acinetobacter baumannii Texas Red Staphylococcus aureus probed using combinatorial FISH for species identiﬁcation. After the hybridization step, cells were imaged in different channels (PhC, Alexa 488, Cy3, Cy5, and Texas Red). The bacterial species are marked in white (Escherichia coli), magenta (Kleb- siella pneumoniae), cyan (Pseudomonas aeruginosa), brown (Enterococcus faecalis), yellow (Acinetobacter baumannii), navy blue (Proteus mirabilis) and red (Staphy- lococcus aureus). The experiment with all seven species mixed was performed a single time. However, similar experiments with all adaptors and probes mixed are reported in Fig. 5. Fig. 4 \| Combinatorial FISH. a Overview of the combinatorial FISH probing for the multi species identiﬁcation. A cartoon illustrating the different bacterial species with their ribosomal RNA (left). Illustration of the speciﬁc sequences with the multiple adapters targeting the ribosomal RNA of individual bacteria and its hybridization to the target rRNA (middle). Detection probes with different ﬂuor- ophores. Hybridization of detection probes to the adapter sequences along with unique sequences that are targeted to the species speciﬁc rRNA (Right). b Example images (Scale bar 20 µm) of mixed species loaded in the microﬂuidic chip and Fig. 4 \| Combinatorial FISH. a Overview of the combinatorial FISH probing for the multi species identiﬁcation. A cartoon illustrating the different bacterial species with their ribosomal RNA (left). Illustration of the speciﬁc sequences with the multiple adapters targeting the ribosomal RNA of individual bacteria and its hybridization to the target rRNA (middle). Detection probes with different ﬂuor- ophores. Hybridization of detection probes to the adapter sequences along with unique sequences that are targeted to the species speciﬁc rRNA (Right). Scaling up FISH probing to 10 species b Example images (Scale bar 20 µm) of mixed species loaded in the microﬂuidic chip and this method to be clinically relevant, it needs to be calibrated and tested on many more clinical isolates as well as on real patient samples. M th d such that they are not mistaken for pathogens. However, if more than 10 speciﬁc ID codes are required, stripping and reprobing allow an exponential increase in how many classes can be identiﬁed35,36, but at the expense of time. Nature Communications\| (2022) 13:6215 https://doi.org/10.1038/s41467-022-33659-1 Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Nitrofurantoin 32μg/ml Nitrofurantoin 32μg/ml Vancomycin 4μg/ml Vancomycin 4μg/ml E. coli (S)Treatment P. mirabilis (S)Treatment No species ID Reference No species ID Treatment E. coli (S)Treatment P. mirabilis (S)Treatment No species ID Reference No species ID Treatment E. coli (R)Treatment E. faecalis (S)Treatment No species ID Reference No species ID Treatment E. coli (R)Treatment E. faecalis (S)Treatment No species ID Reference No species ID Treatment c d g h with Vancomycin. e–h Biological repeats of 5a-5d, respectively. In all AST proﬁle plots, S and R represent Susceptible and Resistance, respectively. Normalized growth rates ± SEM as a function of time are shown for each species detected in the experiments. Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Ciprofloxacin 1μg/ml K. pneumoniae (S) Treatment S. aureus (S) Treatment No species ID Reference No species ID Treatment b Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Vancomycin 4μg/ml E. coli (R)Treatment E. faecalis (S)Treatment No species ID Reference No species ID Treatment d Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Gentamicin 2μg/ml P. aeruginosa (S)Treatment A. baumanni (R)Treatment No species ID Reference No species ID Treatment a Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Nitrofurantoin 32μg/ml E. coli (S)Treatment P. mirabilis (S)Treatment No species ID Reference No species ID Treatment c a Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Gentamicin 2μg/ml P. aeruginosa (S)Treatment A. baumanni (R)Treatment No species ID Reference No species ID Treatment e Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Vancomycin 4μg/ml E. coli (R)Treatment E. Fast phenotypic AST on Antimicrobial Susceptibility Testing (EUCAST). We note that another, higher, concentration may be optimal to make a rapid SIR call. The cells were loaded on the chip and exposed to growth media with and without the antibiotics in two different rows. In each row, a total of 70–80 positions, each including 16–21 cell traps, were images in the phase contrast channel (30 ms exposure time) every two minutes for an hour. Media and culture conditions In all experiments, Mueller-Hinton (MH) medium (70192; Sigma- Aldrich) was used as a broth. Overnight cultures (ONC) were prepared by inoculating bacteria from glycerol stocks (−80 °C) in MH medium and incubating at 37 °C for 14–15 h with continuous shaking (200 rpm). From ONC, cells were diluted 1:1000 times into fresh MH medium supplemented with a surfactant (Pluronic F-108; 542342; Sigma- Aldrich; 0.085% (wt/vol) ﬁnal concentration). The liquid culture was grown by shaking at 200 rpm at 37 °C for 2 h. Next, to perform AST experiments, we mixed the different strains in equal concentrations and loaded them on the microﬂuidic chip. https://doi.org/10.1038/s41467-022-33659-1 https://doi.org/10.1038/s41467-022-33659-1 Article Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Gentamicin 2μg/ml Gentamicin 2μg/ml Ciprofloxacin 1μg/ml Ciprofloxacin 1μg/ml Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Nitrofurantoin 32μg/ml Nitrofurantoin 32μg/ml Vancomycin 4μg/ml Vancomycin 4μg/ml P. aeruginosa (S)Treatment A. baumanni (R)Treatment No species ID Reference No species ID Treatment P. aeruginosa (S)Treatment A. baumanni (R)Treatment No species ID Reference No species ID Treatment K. pneumoniae (S) Treatment S. aureus (S) Treatment No species ID Reference No species ID Treatment K. pneumoniae (S) Treatment S. aureus (S) Treatment No species ID Reference No species ID Treatment E. coli (S)Treatment P. mirabilis (S)Treatment No species ID Reference No species ID Treatment E. coli (S)Treatment P. mirabilis (S)Treatment No species ID Reference No species ID Treatment E. coli (R)Treatment E. faecalis (S)Treatment No species ID Reference No species ID Treatment E. coli (R)Treatment E. faecalis (S)Treatment No species ID Reference No species ID Treatment a b c d e f g h Fig. 5 \| Species-wise AST proﬁles for experiments with 2 species using the combinatorial FISH method. a P. aeruginosa and A. baumannii were treated with Gentamicin, b K. pneumoniae, and S. aureus treated with Ciproﬂoxacin, c E. coli, and P. mirabilis treated with Nitrofurantoin, d E. coli and E. faecalis treated with Vancomycin. e–h Biological repeats of 5a-5d, respectively. In all AST proﬁle plots, S and R represent Susceptible and Resistance, respectively. Normalized growth rates ± SEM as a function of time are shown for each species detected in the experiments. https://doi.org/10.1038/s41467-022-33659-1 faecalis (S)Treatment No species ID Reference No species ID Treatment h 1 f Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Ciprofloxacin 1μg/ml K. pneumoniae (S) Treatment S. aureus (S) Treatment No species ID Reference No species ID Treatment f Growth Rate (normalized) 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 20 30 40 50 Time (min) Nitrofurantoin 32μg/ml E. coli (S)Treatment P. mirabilis (S)Treatment No species ID Reference No species ID Treatment g Ciprofloxacin 1μg/ml Vancomycin 4μg/ml g e Time (min) Fig. 5 \| Species-wise AST proﬁles for experiments with 2 species using the combinatorial FISH method. a P. aeruginosa and A. baumannii were treated with Gentamicin, b K. pneumoniae, and S. aureus treated with Ciproﬂoxacin, c E. coli, and P. mirabilis treated with Nitrofurantoin, d E. coli and E. faecalis treated with Vancomycin. e–h Biological repeats of 5a-5d, respectively. In all AST proﬁle plots, S and R represent Susceptible and Resistance, respectively. Normalized growth rates ± SEM as a function of time are shown for each species detected in the experiments. Genotyping After phenotyping, the medium from ports 2.1 and 2.2 were depres- surized to zero. To ﬁx the cells, formaldehyde (4%) was added by switching the medium and applying pressure 200 mbar from port 2.0 for 4 min and subsequently washing the cells with 1 × phosphate- buffered saline (PBS) for 3 min. Cells were permeabilized by 70% EtOH for 4 min and washed with 1 × PBS (3 min). Cells were next treated with lysozyme (2 mg/ml) and lysostaphin (0.1 mg/ml) for 3 min and fol- lowed by quick washing with 1 × PBS for another 3 min. (Lysostaphin is only needed for S. aureus and was not used in Fig. 3). For species identiﬁcation, we pooled all speciﬁc ssDNA probes (0.1 μM) (Supple- mentary Data 1) in a hybridization solution (30% formamide and 2 × SSC) and hybridized them for 30 min at 30 °C. In the case of the combinatorial method, we pooled all the detection probes (0.1 μM) (Supplementary Data 2) and species-speciﬁc adaptor sequences (Supplementary Data 3) in a hybridization solution (30% formamide and 2 × SSC) and hybridized them for 60 min at 30 °C. Next, we cap- tured the ﬂuorescence images for each probe in different channels (TYE 665, TYE 563, Texas Red, and Alexa Fluor 488) at 300 ms expo- sure times and respective phase contrast images at 30 ms exposure time. In total, it took 10–15 min to image all the positions in all the channels on the chip. The microﬂuidic chip and setup The chip consist of mainly two parts: a micromolded silicone elasto- mer [Sylgard 184; polydimethylsiloxane (PDMS)] and a 1.5 glass cov- erslip (Menzel-Gläser) which are covalently bonded together. Chip design and preparation were previously described in15,38. These refer- ences also describe the numbering of the ports used below. After punching the ports on the chip, it was placed on the microscope, and tubing (TYGON) was connected with a metal tubing connector. Brieﬂy, cells were loaded using port 8.0 and port 2.0 was used for the exchange of medium with the probes. Ports 5.1, 5.2, and 6.0 were used for maintenance of back-channel pressure, and ports 2.1 and 2.2 were used for the supply of MH medium with and without the antibiotics at a pressure of 200 mbar. The pressure was controlled by an OB1-Mk3 regulator (Elveﬂow). Bacterial strains and antibiotics In the current implementation, we ran the test one concentration at a time on a high-end research microscope and post-processed the imaging data. To make the technology useful in a clinical setting, data should be analyzed during the experiment and the ﬂuidic chip should be parallelized to run multiple antibiotic concentrations simulta- neously. Although we have not tested this type of setup in the present study, it should allow a MIC determination to identify intermediate isolates. A system with ﬁve different antibiotics at ﬁve different con- centrations and a high level of automation has been developed for uncomplicated urinary tract infections20, which shows that upscaling to multiple testing conditions is possible. Finally, we would like to emphasize that the present study is proof of principle of combined phenotypic AST and species identiﬁcation at the single-cell level. For In this study, as a gram-negative representative we used E. coli K12 MG1655 (DA4201), K. pneumoniae (ATCC 13883), A. baumanni (DA68153), P. mirabilis (ATCC 29906) and P. aeruginosa (DA6215). As a gram-positive representative, we used E. faecalis (ATCC 51299) and S. aureus (ATCC 29213). We also used the ﬂuorescently tagged P. aeru- ginosa (PAO1-GFP) cells, a kind gift from Oana Ciofu37. Antibiotics (Ciproﬂoxacin, Gentamicin, Nitrofurantoin, and Vancomycin) were purchased from Sigma-Aldrich. Stock solutions were prepared as per the supplier guidelines and stored at −20 °C. The solutions were thawed to room temperature before performing the AST experiments. We used concentrations of the antibiotics corresponding to the MIC values for E. coli (ATCC 25922) as deﬁned by the European Committee Nature Communications\| (2022) 13:6215 5 Reporting summary 17. Rychert, J. Beneﬁts and limitations of MALDI-TOF mass spectro- metry for the identiﬁcation of microorganisms. J. Infectiology 2, 1–5 (2019). Further information on research design is available in the Nature Research Reporting Summary linked to this article. 18. Ferreira, L. et al. Direct identiﬁcation of urinary tract pathogens from urine samples by matrix-assisted laser desorption ionization-time of ﬂight mass spectrometry. J. Clin. Microbiol. 48, 2110–2115 (2010). References 1. GBD 2016 Causes of Death Collaborators Global, regional, and national age-sex speciﬁc mortality for 264 causes of death, 1980–2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet 390, 1151–1210 (2017). 1. GBD 2016 Causes of Death Collaborators Global, regional, and national age-sex speciﬁc mortality for 264 causes of death, 1980–2016: A systematic analysis for the Global Burden of Disease Study 2016. Lancet 390, 1151–1210 (2017). Oligos and probes design FISH probe sequences for the individual target rRNA were obtained from the probeBase21,40,41 and purchased from Integrated DNA Technologies (www.idt.com), see Supplementary data 1. For the combinatorial FISH probing, we used barcode sequence and detection probes, which are listed in Supplementary Data 2–4, purchased from IDT. 11. Ventola, C. L. The antibiotic resistance crisis: part 1: Causes and threats. P T 40, 277–283 (2015). 12. Vasala, A., Hytönen, V. P. & Laitinen, O. H. Modern tools for rapid diagnostics of antimicrobial resistance. Front. Cell. Infect. Microbiol. 10, 308 (2020). 13. Cockerill, F. R. Genetic methods for assessing antimicrobial resis- tance. Antimicrobial Agents Chemother. 43, 199–212 (1999). Microﬂuidic experiments Phase-Contrast images of the cells growing in channels were seg- mented for both cells and channels using a deep learning model with Omnipose method. The cell-segmentation model was trained with data obtained from manually-labeled images of mixed species data and data obtained from E. coli (K12 MG1655 intC::P70-venusFast) that con- stitutively expresses mVenus. The training procedure was enhanced with data augmentations to force learning outputs of Omnipose net- work in different orientations and scales. The model training and performance compared to U-net is described in SI method 1. The Imaging starts within ﬁve minutes after the supply of medium with and without the antibiotics to the cells. We used a Nikon Ti2-E inverted microscope equipped with a Plan Apo Lambda 100× oil immersion objective (Nikon). Images were captured by the Imaging Source (DMK 38UX304) camera. For phase contrast and ﬂuorescence images, we used the optical setup as previously described in15,38 and controlled by Micro-Manager39, and an in-house built plugin. We maintained 30 °C using a temperature controllable unit and a lexan enclosure (Oklab). Nature Communications\| (2022) 13:6215 6 Article https://doi.org/10.1038/s41467-022-33659-1 Code availability segmentation network performance on different species is also shown in SI method 1. The channel segmentation model was trained with data that was reﬁned based on histogram proﬁles, also described in SI method 2. After obtaining channel locations, time-series stacks of segmented cells and corresponding ﬂuorescent channel images were bundled for tracking, species assignment, and growth rate calculations. The code used for analysis and reproducing all the ﬁgures is available at https://github.com/karempudi/ASTFISH.git. Weights of trained neural networks for cell segmentation and tracking are available at https://doi.org/10.17044/scilifelab.20969161. Cell tracking Cells were tracked through time using a neural network that scores links between cells from one frame to the next and predicts the type of link between them. Training data for cell tracking was obtained by simulating the growth of cells in mother-machine channels and is described in SI method 4. Cells of different shapes, sizes, and growth rates were used to generate ground truths for the tracking network. Tracker network training and its performance are described in SI method 4. The predictions of the network are cleaned for error using IoU (Intersection-over-Union) metric to remove spurious predictions. 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Cell tracks falling in these regions in the last frame before ﬁxing cells were labeled with the corresponding species and all the species labels were rolled back to time point 0. Growth rates were calculated by ﬁtting exponential curves on the areas of cells in a moving 5 timepoint window (SI method 7). 7. Kumar, A. et al. Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. Crit. Care Med. 34, 1589–1596 (2006). 8. Ibrahim, E. H., Sherman, G., Ward, S., Fraser, V. J. & Kollef, M. H. The inﬂuence of inadequate antimicrobial treatment of bloodstream infections on patient outcomes in the ICU setting. Chest 118, 146–155 (2000). 9. Caliendo, A. M. et al. Better tests, better care: Improved diagnostics for infectious diseases. Clin. Infect. Dis. 57, S139–S170 (2013). 10. Kumar, A. et al. Initiation of inappropriate antimicrobial therapy results in a ﬁvefold reduction of survival in human septic shock. 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https://openalex.org/W4283398085	https://www.frontiersin.org/articles/10.3389/fcvm.2022.916096/pdf	English	null	Case Report: A Rare Abdominopelvic Arteriovenous Malformation: Originating From Splenic Artery and Draining Into Portal Vein	Frontiers in cardiovascular medicine	2,022	cc-by	3,459	CASE REPORT published: 23 June 2022 doi: 10.3389/fcvm.2022.916096 CASE REPORT published: 23 June 2022 doi: 10.3389/fcvm.2022.916096 CASE REPORT Case Report: A Rare Abdominopelvic Arteriovenous Malformation: Originating From Splenic Artery and Draining Into Portal Vein Xin Li 1†, Jiehua Li 1†, Mo Wang 1, Junwei Wang 1, Lunchang Wang 1, Hao He 1, Ming Li 1, Quanming Li 1 and Chang Shu 1,2* Xin Li 1†, Jiehua Li 1†, Mo Wang 1, Junwei Wang 1, Lunchang Wang 1, Hao He 1, Ming Li 1, Quanming Li 1 and Chang Shu 1,2* 1 Department of Vascular Surgery, The Second Xiangya Hospital, Central South University, Changsha, China, 2 Center of Vascular Surgery, Chinese Academy of Medical Sciences and Peking Union Medical College, Fuwai Hospital, Beijing, China Background: Abdominopelvic arteriovenous malformation is an uncommon congenital vascular lesion, for which the diagnosis and treatment are usually difﬁcult. Though embolization and sclerotherapy are the primary treatment strategies, traditional surgical resection remains a valuable option. Reviewed by: Su Li Xin, Reviewed by: Su Li Xin, Shanghai Jiao Tong University, China Hai Feng, Capital Medical University China Su Li Xin, Shanghai Jiao Tong University, China Hai Feng, Conclusion: To our knowledge, the presented abdominopelvic arteriovenous malformation is the ﬁrst to be reported in the literature, with such a rare condition originating from the splenic artery and draining into the portal vein. Correspondence: Chang Shu shuchang@csu.edu.cn Keywords: abdominopelvic arteriovenous malformation, vascular malformation, splenic artery, portal vein, surgical resection †These authors have contributed equally to this work and share ﬁrst authorship BACKGROUND Specialty section: This article was submitted to Cardiovascular Imaging, a section of the journal Frontiers in Cardiovascular Medicine Received: 08 April 2022 Accepted: 25 May 2022 Published: 23 June 2022 Specialty section: This article was submitted to Cardiovascular Imaging, a section of the journal Frontiers in Cardiovascular Medicine Abdominopelvic arteriovenous malformation (AVM) is an uncommon vascular lesion, for which the etiology is usually congenital, and the diagnosis and treatment are diﬃcult. The AVM is characterized by abnormal connections between supplying arteries and draining veins (1). It may begin with an asymptomatic lesion, but often progresses over time and expands secondary to trauma or hormonal change, such as that during puberty or pregnancy (2). The symptoms range from mild discomfort to high-output cardiac failure or life-threatening hemorrhage (3). Though embolization and sclerotherapy are the primary treatment strategies, traditional surgical resection remains a valuable option (4–6). Case Presentation: Herein, we present a 32-year-old female diagnosed with a massive abdominopelvic arteriovenous malformation that originates from the splenic artery and drains into the portal vein. The vascular lesion was evaluated with multiple imaging modalities and then surgically resected successfully. The patient was discharged post-operatively on day 6 and free of symptoms during the 12-month follow-up. Edited by: Yueqi Zhu, Shanghai Jiao Tong University, China Reviewed by: Su Li Xin, Shanghai Jiao Tong University, China Hai Feng, Capital Medical University, China Correspondence: Chang Shu shuchang@csu.edu.cn Edited by: Y i Zh Edited by: Yueqi Zhu, Shanghai Jiao Tong University, China Edited by: Yueqi Zhu, Shanghai Jiao Tong University, China Shanghai Jiao Tong University, China Citation: Most pelvic AVMs are supplied by internal iliac arteries, inferior mesenteric artery, or the median sacral artery (7–9), and are often drained into iliac, pudendal, or obturator veins (10–12). However, for the current case we present here, the AVM rarely had a single feeding artery from the splenic artery and two draining veins into the portal vein, which, to our knowledge, has not been reported before. The patient was asymptomatic and physical examination revealed aberrant abdominal vascular bruits. The massive abdominopelvic AVM was evaluated with multiple imaging modalities and successfully treated with surgical resection. Li X, Li J, Wang M, Wang J, Wang L, He H, Li M, Li Q and Shu C (2022) Case Report: A Rare Abdominopelvic Arteriovenous Malformation: Originating From Splenic Artery and Draining Into Portal Vein. Front. Cardiovasc. Med. 9:916096. doi: 10.3389/fcvm.2022.916096 June 2022 \| Volume 9 \| Article 916096 Frontiers in Cardiovascular Medicine \| www.frontiersin.org Li et al. Treatment of a Rare AVM FIGURE 1 \| Evaluation of the abdominopelvic arteriovenous malformation (AVM) with color Doppler ultrasound and CT angiography. (A) The ultrasound revealed a pelvic mass with an abundant blood ﬂow signal. (B) The axial view of CT angiography showed the nidus of AVM (yellow arrow) and dilated outﬂow veins (white arrows) (the aneurysm was measured as 72 × 56 mm). FIGURE 1 \| Evaluation of the abdominopelvic arteriovenous malformation (AVM) with color Doppler ultrasound and CT angiography. (A) The ultrasound revealed a pelvic mass with an abundant blood ﬂow signal. (B) The axial view of CT angiography showed the nidus of AVM (yellow arrow) and dilated outﬂow veins (white arrows) (the aneurysm was measured as 72 × 56 mm). FIGURE 2 \| 3D reconstruction of CT angiography of the abdominopelvic arteriovenous malformation (AVM). (A) Arterial phase of CT angiography showed the AVM had a single feeding artery originating from the splenic artery. (B) Venous phase of CT angiography showed the AVM had two draining veins into the superior mesenteric vein and then the portal vein. lf i (AVM) (A) A i l h f CT i h h d h AVM FIGURE 2 \| 3D reconstruction of CT angiography of the abdominopelvic arteriovenous malformation (AVM). (A) Arterial phase of CT angiography showed the AVM had a single feeding artery originating from the splenic artery. Citation: (B) Venous phase of CT angiography showed the AVM had two draining veins into the superior mesenteric vein and then the portal vein. June 2022 \| Volume 9 \| Article 916096 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 2 Li et al. Treatment of a Rare AVM FIGURE 3 \| Digital subtraction angiography (DSA) and resected vascular lesion of the abdominopelvic arteriovenous malformation (AVM). (A) DSA showed the feeding artery and AVM nidus. (B) DSA showed the AVM nidus and two draining veins. (C) DSA showed draining veins ﬂowed into the superior mesenteric vein and then the portal vein. (D) the AVM lesion was surgically removed successfully. CASE PRESENTATION On physical examination her mass was not palpable but vascular FIGURE 3 \| Digital subtraction angiography (DSA) and resected vascular lesion of the abdominopelvic arteriovenous malformation (AVM). (A) DSA showed the feeding artery and AVM nidus. (B) DSA showed the AVM nidus and two draining veins. (C) DSA showed draining veins ﬂowed into the superior mesenteric vein and then the portal vein. (D) the AVM lesion was surgically removed successfully. DISCUSSION AND CONCLUSION (AVM) and aneurysmal-dilated vessel (measured 72 × 56 mm) (Figure 1B). The 3D reconstruction of CTA demonstrated that the AVM had a single feeding artery originating from the splenic artery and two draining veins into the superior mesenteric vein, and then, a portal vein (Figure 2). Digital subtraction angiography (DSA) was used to further evaluate the AVM (Figures 3A–C). It was conﬁrmed that the AVM ﬁnally drained into the portal vein and there were no other feeding arteries from bilateral internal iliac arteries (Supplemenatry Video 1). Considering the risk of aneurysm rupture and hemorrhage, the patient was treated with surgical resection of the AVM and ligation of the feeding artery and draining veins (Figure 3D). She recovered well after the operation and was discharged post- operatively on day 6. The 1-month follow-up CTA showed elimination of the abdominopelvic AVM (Figure 4) and the patient was doing well during the following 12 months. (AVM) and aneurysmal-dilated vessel (measured 72 × 56 mm) (Figure 1B). The 3D reconstruction of CTA demonstrated that the AVM had a single feeding artery originating from the splenic artery and two draining veins into the superior mesenteric vein, and then, a portal vein (Figure 2). Digital subtraction angiography (DSA) was used to further evaluate the AVM (Figures 3A–C). It was conﬁrmed that the AVM ﬁnally drained into the portal vein and there were no other feeding arteries from bilateral internal iliac arteries (Supplemenatry Video 1). Considering the risk of aneurysm rupture and hemorrhage, the patient was treated with surgical resection of the AVM and ligation of the feeding artery and draining veins (Figure 3D). She recovered well after the operation and was discharged post- operatively on day 6. The 1-month follow-up CTA showed elimination of the abdominopelvic AVM (Figure 4) and the patient was doing well during the following 12 months. Arteriovenous malformation (AVM) belongs to the entity of vascular malformations, which are generally classiﬁed into slow- ﬂow (venous, capillary, and lymphatic malformation) and fast- ﬂow malformations (arteriovenous ﬁstula and arteriovenous malformation) (13). AVM is an uncommon medical condition and accounts for 8% of vascular malformations (14). It could appear on almost any part of the body, including the abdominal and pelvic cavity (15). The composition of a typical AVM includes feeding arteries, draining veins, and the vascular shunting bypassing the capillary bed (also called “the nidus”) (3). CASE PRESENTATION On physical examination, her mass was not palpable, but vascular bruits could be easily auscultated on the abdomen, especially in the peri-umbilicus area. The ultrasound revealed a pelvic mass with an abundant blood ﬂow signal (Figure 1A). CT angiography (CTA) showed the nidus of pelvic arteriovenous malformation A 32-year-old woman was admitted to our center due to the ﬁnding of a pelvic mass with hypervascularity 2 years ago. She was generally asymptomatic and had no related family history. June 2022 \| Volume 9 \| Article 916096 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 3 Li et al. Treatment of a Rare AVM FIGURE 4 \| One-month post-operative CT angiography of the patient. Arterial phase (A), Venous phase (B), and axial view (C) of the CT angiography showed elimination of the abdominopelvic AVM. FIGURE 4 \| O th t ti CT i h f th ti t A t i l h FIGURE 4 \| One-month post-operative CT angiography of the patient. Arterial phase (A), Venous phase (B), and axial view (C) of the CT angiography showed li i ti f th bd i l i AVM FIGURE 4 \| One-month post-operative CT angiography of the patient. Arterial phase (A), Venous phase (B), and axial view (C) of the CT angiography showed elimination of the abdominopelvic AVM. erative CT angiography of the patient. Arterial phase (A), Venous phase (B), and axial view (C) of the CT angiography showed Frontiers in Cardiovascular Medicine \| www.frontiersin.org DISCUSSION AND CONCLUSION Speciﬁcally for our case, the AVM had a feeding artery from the splenic artery, two draining veins into the portal vein, and signiﬁcant dilated vascular “nidus” formed in the pelvic cavity. June 2022 \| Volume 9 \| Article 916096 Frontiers in Cardiovascular Medicine \| www.frontiersin.org 4 Li et al. Treatment of a Rare AVM the pelvic wall. For this case, surgical resection was considered to be a superior strategy to endovascular therapy. As the AVM had relatively simple angioarchitectures without involving vital adjacent vital organs, surgical resection could provide a deﬁnite and eﬀective treatment, while interventional therapy had the risk of incomplete or ectopic embolization. Pelvic AVM may be asymptomatic in some patients, and present as a pulsatile or vascularized mass on pelvic imaging. However, AVMs often progress throughout life, and a variety of symptoms could occur, such as urinary frequency or dysuria, pelvic pain, life-threatening hemorrhage, and high-output cardiac failure. It is well-known that the growth of AVM could be stimulated by various factors, such as puberty, pregnancy, and trauma. According to the Schobinger classiﬁcation system, there are typically four stages for AVM lesions: stage I (quiescent), stage II (expansive), stage III (destructive), and stage IV (cardiac decompensation) (16). The current case had a stage II pelvic AVM that was asymptomatic, but with a signiﬁcantly dilated vascular lesion. She presented no signs of heart failure or portal hypertension. The patient was treated successfully with surgical resection of the AVM and ligation of the feeding artery and draining veins. A major concern of the surgery was that removal of the AVM might signiﬁcantly decrease the blood ﬂow into the liver and cause ischemic liver injury. Fortunately, the patient recovered well and showed no signs of liver injury after the operation. The post-operative CTA showed no evidence of residual arteriovenous connections, and she lived uneventfully during the 12-month follow-up. The pelvic AVMs are usually ﬁrst detected and evaluated by color Doppler ultrasound, which could oﬀer an impressive overview of the lesion with hypervascularity signals (17). MR angiography and CT angiography are important diagnostic tools to visualize the feeding arteries, draining veins, and location of shunting, as well as ﬂow dynamics (16). However, digital subtraction angiography (DSA) remains a pre-requisite for the treatment of AVMs. DSA via transarterial, transvenous, and sometimes direct percutaneous access could profoundly evaluate the AVM. SUPPLEMENTARY MATERIAL Here we presented a condition of AVM that might not have been described in the literature before, which had a massive vascular nidus in the pelvic cavity with feeding artery from splenic artery and draining veins into portal vein. The AVM was profoundly evaluated with color Doppler ultrasound, CT angiography, and catheter angiography, and it was demonstrated as a well-localized lesion that did not involve other organs or The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm. 2022.916096/full#supplementary-material Supplementary Video \| Digital subtraction angiography of the massive abdominopelvic arteriovenous malformation. Supplementary Video \| Digital subtraction angiography of the massive abdominopelvic arteriovenous malformation. DISCUSSION AND CONCLUSION The successful treatment of AVM depends largely on the diagnostic imaging, which needs to provide information on the location, extension, composition, vessel diameters, and hemodynamics of AVM. FUNDING This work was supported by the National Natural Science Foundation of China (grant numbers: 81870345 and 82120108005) and the Natural Science Foundation of Hunan Province, China (grant number: 2020JJ2054). This work was supported by the National Natural Science Foundation of China (grant numbers: 81870345 and 82120108005) and the Natural Science Foundation of Hunan Province, China (grant number: 2020JJ2054). ETHICS STATEMENT The studies involving human participants were reviewed and approved by the institutional Ethics boards of the Second Xiangya Hospital. The patients/participants provided their written informed consent to participate in this study. Treatment of AVM is notoriously challenging, as inappropriate treatment could stimulate the AVM lesion into an aggressive growth state (18). Although an asymptomatic lesion can be observed, it is preferable to treat the AVM before it progresses. Surgical resection of AVM lesions is an eﬀective strategy and has been regarded as the gold standard for a long period, but it carries the risk of massive bleeding, incomplete resection of the lesion, and injury to adjacent organs. In recent decades, endovascular therapy using various embolic and sclerosing materials, either alone or in a combination with surgical resection, has become a widely accepted treatment option for patients with AVMs (19, 20). However, the endovascular strategy could still lead to signiﬁcant recurrence and complication rate (21). Thus, the optimal treatment of AVM should be individualized and be based on the certain condition of AVM and the experience of surgeons. AUTHOR CONTRIBUTIONS XL and JL drafted the manuscript. CS and QL designed the study. HH and ML revised the manuscript. MW, JW, and LW were responsible for the collection of data or analysis. All authors read and approved the ﬁnal manuscript. 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https://openalex.org/W3117820265	http://umu.diva-portal.org/smash/get/diva2:1517816/FULLTEXT02	English	null	Online and offline sexual harassment associations of anxiety and depression in an adolescent sample	Nordic journal of psychiatry	2,020	cc-by	5,691	Nordic Journal of Psychiatry Nordic Journal of Psychiatry ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ipsc20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ipsc20 ABSTRACT ABSTRACT Purpose: The aims of this study were to study the prevalence of sexual harassment online and offline, to analyse the associations between subjection to sexual harassment and adolescents’ mental health and analyse if there are any significant differences between girls and boys. We also examine if good peer-relationships interact with the associations between sexual harassment and mental health complaints. KEYWORDS Adolescents; offline; online; sexual harassment Methods: This cross-sectional study included 594 adolescents, age 12–20. Participants responded to a web survey including the self-assessment scales Revised Childrens Anxiety and Depression Scale and Beck Youth Inventories as well as subjection to online and offline sexual harassment and peer-rela- tional quality. Linear regression analysis was used to study whether symptoms of anxiety and depres- sion correlated to subjection to online and offline sexual harassment and peer-relational quality. Results: We found that 48.50% of girls and 28.19% of boys reported sexual harassment victimization. Offline was the most frequently reported site of victimization. Online harassment correlated signifi- cantly with increased anxiety and depressive symptoms in girls but not boys. Offline harassment as well as online and offline harassment correlated significantly with increasing symptoms for both gen- ders. Participants who reported good peer-relationships had significantly less symptoms. Conclusions: This study shows that sexual harassment remains a common plague for adolescents, especially for girls. Offline sexual harassment is the most common form of harassment for both gen- ders. For girls, but not for boys, online sexual harassment correlated significantly with anxiety and depressive symptoms. A strong negative correlation between satisfaction to peer-relationships and mental health symptoms was found. Online and offline sexual harassment associations of anxiety and depression in an adolescent sample Simon Ståhl & Inga Dennhag To cite this article: Simon Ståhl & Inga Dennhag (2021) Online and offline sexual harassment associations of anxiety and depression in an adolescent sample, Nordic Journal of Psychiatry, 75:5, 330-335, DOI: 10.1080/08039488.2020.1856924 To link to this article: https://doi.org/10.1080/08039488.2020.1856924 © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 21 Dec 2020. Submit your article to this journal Article views: 1831 View related articles View Crossmark data Citing articles: 3 View citing articles © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 21 Dec 2020. Submit your article to this journal Article views: 1831 View related articles View Crossmark data Citing articles: 3 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ipsc20 NORDIC JOURNAL OF PSYCHIATRY 2021, VOL. 75, NO. 5, 330–335 https://doi.org/10.1080/08039488.2020.1856924 NORDIC JOURNAL OF PSYCHIATRY 2021, VOL. 75, NO. 5, 330–335 https://doi.org/10.1080/08039488.2020.1856924 Online and offline sexual harassment associations of anxiety and depression in an adolescent sample Simon Ståhla and Inga Dennhagb aFaculty of Medicine Child- and Adolescent Psychiatry, Umeå University, Umeå, Sweden; bDepartment of Clinical Science, Child- and Adolescent Psychiatry, Umeå University, Umeå, Sweden ARTICLE HISTORY Received 6 May 2020 Revised 15 November 2020 Accepted 23 November 2020 ARTICLE HISTORY Received 6 May 2020 Revised 15 November 2020 Accepted 23 November 2020 KEYWORDS Adolescents; offline; online; sexual harassment p g 2020 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. Faculty of Medicine Child- and Adolescent Psychiatry, Umeå University, Fj€arilsv€agen 132, 587 ma UK Limited, trading as Taylor & Francis Group. under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, um, provided the original work is properly cited. Introduction sexual victimization whereas online sexual solicitations were reported in 35.5% of girls and 19.9% of boys [3]. In a country- wide Finnish study, 55% of girls and 40% of boys reported sex- ual victimization [4]. Two cross-sectional studies from Norway found prevalence numbers of 80% among girls and 74% among boys in 2007, and 64% and 62%, respectively, in 2013 [5]. An American study found a lower prevalence at 29% and 20% for girls and boys respectively [6]. Given this variation, local studies are important to accurately estimate the actual preva- lence of this problem in different countries. Reports about the occurrence of sexual harassment in different areas of society have succeeded each other in recent years. Sexual harassment can take many forms but is usually divided into physical, verbal, or non-verbal forms and can take place in person (offline) or through media such as the internet or phone (online) [1]. Previous studies often have not separated harass- ment that takes place online from harassment offline. Given the fact that smartphone access and internet use are nearly univer- sal among Swedish teens [2], there is a need to understand the impact online experiences have on the mental health of adoles- cents. Previous studies of sexual harassment, online and offline, among adolescents have found that the prevalence is relatively large, but this varies among studies from different countries, the year of measurement, and the definition of sexual harass- ment. Different studies use different strategies to ask for the respondent experiences; while some provide examples of situa- tions of harassment for the respondent to answer, others ask open questions where the respondents themselves are left to think what situations to include. A previous Swedish study found that 51.6% of girls and 45.1% of boys reported offline Some studies have found that sexual harassment victim- ization correlates with symptoms of anxiety in adolescent girls and boys [5], whereas other studies have found this cor- relation in girls but not in boys [7]. Cross-sectional data has linked sexual harassment victimization to other forms of mental illness outcomes among adolescents, such as higher levels of self-harm [6,8], substance use and depressive symp- toms [6]. NTACT Simon Ståhl simon.staahl88@gmail.com Faculty of Medicine Child- and Adolescent Psychiatry, Umeå University, Fj€arilsv€agen 132, 587 Link€oping, Sweden 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. CONTACT Simon Ståhl simon.staahl88@gmail.com Faculty of Medicine Child- and Adolescent Psychiatry, Umeå University, Fj€arilsv€agen 132, 587 29 Link€oping, Sweden 2020 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. aahl88@gmail.com Faculty of Medicine Child- and Adolescent Psychiatry, Umeå University, Fj€arilsv€agen 132, 587 CONTACT Simon Ståhl simon.staahl88@gmail.com Faculty of Medicine Child- and Adolescent Psychiatry, Umeå University, Fj€arilsv€agen 132, 587 29 Link€oping, Sweden Introduction Furthermore, longitudinal data shows that partici- pants who report more sexual harassment victimization at one point were more likely to report more emotional distress and symptoms of negative affect on the following measures, CONTACT Simon Ståhl 29 Link€oping, Sweden simon.staahl88@gmail.com CONTACT Simon Ståhl 29 Link€oping, Sweden NORDIC JOURNAL OF PSYCHIATRY 331 indicating a cause-and-effect relation [8,9]. Contrary to the above findings, however, a recent Finnish study found that subjection to sexual harassment was inversely or non-signifi- cantly associated with depressive symptoms in adolescents. Although a significant correlation was found with social anx- iety [7]. Considering this somewhat surprising result, we found it important to include both depressive and anxiety assessments in the present analysis. purpose of that project was to develop new instruments for screening and treating mental illness among young patients. Ethical approval was given by the Regional Ethical Review Board in Umeå, Sweden (number 2018/59-31). The study was conducted in a medium-sized town and its surroundings in the northern part of Sweden. Data collection took place dur- ing the school year 2018–2019 and was spread over most school months that year. Internet and technological advances have opened new ways for communication and social participation among people [10]. As more social interactions take place online, the potential risk of cyber victimization increases. A national survey in Sweden in 2019 showed that all teenagers use the internet daily, and nearly all possess a smartphone [2]. Parallel to this, the number of teenagers who have reported being the victims of bullying and sexual assaults online has increased in recent years [2], which is a shift in trend from previous years [11]. A number of studies have found that girls are more frequently the victims of sexual harassment both online and offline [12–14]. However, not all studies are aligned. A meta-analysis of the prevalence of unwanted online sexual solicitation from 1990 to 2016 found an overall decrease since 2000, and boys were more frequently the victims [11]. A Swedish study found that online sexual har- assment was associated with a higher level of depressive symp- toms in girls. The association was non-significant for boys when adjusting for social support and offline sexual harassment. The same study also found that offline sexual harassment predicted online sexual harassment, indicating that victims are often tar- geted both online and offline [3]. Participants The study population was a convenience sample containing students from grades 7–9, age 12–16, and gymnasium grade 1–3, age 17–20 (M ¼ 15.73, SD ¼ 1.77). A total of 897 stu- dents were asked to participate in the study and 617 (69%) agreed to do so. 594 respondents were included in the anal- yses after the removal of respondents with too much missing data, 367 girls and 227 boys. Parallel to doing research on the predictors of mental ill- ness, attention must also be given to possible factors allevi- ating the symptoms. Among other factors, peer support has been associated with lower levels of depressive and anxiety symptoms in adolescents [15]. Low peer support has been found to increase the odds of suffering depressive symptoms [3]. Therefore, we find it important to examine if experienc- ing good peer relationships is linked to a lesser likelihood of reporting sexual harassment victimization. Procedure Four schools from areas of different socioeconomic standards were included after permission was granted by their princi- pals. Verbal and written information was given to students by their teachers, written consent was then obtained from the students who chose to volunteer. Informed consent was obtained from the parents of students younger than 15 years of age. Each participant received a login code to access the web survey. Time to fill out the web survey was given during school hours. All questions had to be answered to finish the survey, therefore no missing data was meant to be created. Due to a human mistake, for some students it was possible to complete the survey without all questions answered ren- dering some missing data. A teacher and research assistant were present to answer questions from the students during their participation. Participants were informed that they could withdraw their participation at any time without giving any reason. A snack was provided during participation and a cinema gift card was given to participants after completing the questionnaire. Introduction Despite the integration of technology into everyday life [2], few studies have analysed the effects of sexual victimization online separately from sexual vic- timization offline [3,12]. As victimization online has increased and usage is universal, it is important to examine whether the negative impact on young peoples mental health is similar to traditional forms of peer victimization offline. Dependent variables In summary, the aims of this study are to: (1) Study the prevalence of sexual harassment online and offline. (2) Analyse the associations between subjection to sexual harassment and adolescents mental health. (3) Explore if there are any differences between girls and boys. (4) Explore if satisfaction with peer-relationships interacts with the associations between sexual harassment and mental health complaints. Dependent variables Revised Children’s Anxiety and Depression Scale (RCADS). The scale contains 47 items, of which 37 measure symptoms of anxiety and 10 measure symptoms of depression. Due to a mistake when programming the web survey some ques- tions were not collected. To minimize the impact of this error, the depression scale was not used. Participants with too many missing data points were excluded. Answers were graded on a four-grade Likert scale ranging from never (0) to always (3). Respondents with data missing on more than 10 items for the whole anxiety scale, or more than two items on a separate sub-scale, were not included in the analyses [16]. The RCADS scale has shown a very good internal con- sistency with Cronbach’s alpha of 0.93 across many different countries and languages [17]. RCADS has also shown good Anxiety and depressive symptoms Gender. The questionnaire contained one question where participants declared their legal gender. Girls, on average, reported higher levels of anxiety and depressive -symptoms, assessed with RCADS and BYI-D, than boys. Mean values differed significantly (p < 0.001) between girls and boys for both scales (RCADS: girls [M ¼ 32.46, SD ¼ 18.53]; boys [M ¼ 19.18, SD ¼ 16.08]. BYI-D: girls [M ¼ 17.93, SD ¼ 11.20]; boys [M ¼ 13.09, SD ¼ 10.31].) Satisfaction with peer relationships: The questionnaire contained a three-question-form about participants’ relation to peers. The questions were: ‘has it been easy for you to make new friends and then stay friends with them,’ ‘do you feel that other peers want to be friends with you,’ and ‘are you satisfied with your relations to your friends?’ Answers were graded on a five-grade Likert scale ranging from ‘never’ to ‘almost always.’ All participants’ answers were graded and averaged, generating a score from one to five, corresponding to their overall peer-relational quality. Prevalence of sexual harassment Offline sexual harassment was the most common type of harassment for both genders. It was more common for girls to be victimized both online and offline than for boys. Similarly, fewer girls than boys reported they had never been harassed. No other statistical differences were found. See Table 1. Independent variables ffl d l Independent variables Offline and online sexual harassment. Questions about sexual harassment experiences were inspired by previously used questionnaires [21]. Participants were asked to think back on the previous six months and report any experience of verbal, physical, or non-verbal sexual harassment. Examples were given for each sub-category: (1) Examples of verbal harass- ment: being called a fag or pussy against your will, or some- one commenting on your private life in a sexual manner. (2) Examples of physical harassment: against your will, someone trying to grab you in a sexual manner, pulling on your clothes; for example, underwear or bra-strap. 3. Examples of non-verbal harassment: if someone spreads sexual rumours, sends pictures, messages, or films against your will. Questions for each sub-category were answered by choosing ‘no, never’; ‘yes, at school’; ‘yes, through the internet or phone’; or ‘yes, elsewhere’. The option ‘yes, through the internet or phone’ was defined as online sexual harassment and options ‘yes, at school’ and/or ‘yes, elsewhere’ were defined as offline harassment. Based on the answers from all three questions, each student was grouped into one of four groups (Online, Offline, Online and offline and Not harassed). From these groups, four dichotomous variables for harass- ment were created (‘online,’ ‘offline,’ ‘online and offline,’ and ‘not harassed’) and used for analysis. Tests of assumptions were calculated. Continuous varia- bles were checked for normality using histograms and scores for skewness and kurtosis. RCADS-, BYI-D, and peer-relation -scores were found skewed, however since the sample size was large, violations of this assumption were tolerated [23]. Residual P-plots and scatterplots indicated that the assump- tions of linearity and homoscedasticity were satisfied. Independent variables showed no significant multicollinear- ity, with no Variance Inflation Factor (VIF) above 2. Statistical analyses reliability for cross-cultural use [17]. Cronbachs alpha was 0.95 (95% CI [0.93 0.96]) in the present study. RCADS has been shown to be a valid instrument with moderate to good correlations with other anxiety assessment instruments for non-clinical samples [18]. reliability for cross-cultural use [17]. Cronbachs alpha was 0.95 (95% CI [0.93 0.96]) in the present study. RCADS has been shown to be a valid instrument with moderate to good correlations with other anxiety assessment instruments for non-clinical samples [18]. All statistical analyses were conducted using SPSS, Version 26.0. Gender differences in the prevalence of sexual harass- ment victimization were analysed using the Chi-square test and Phi-coefficients. Gender differences in mean scores for dependent variables were compared using Independent Sample t-Tests. Effect sizes were small using the definition proposed by Cohen [22], with Phi-Coefficients of 0.10 for small effects, 0.30 for medium effects, and 0.50 for large effects. Linear multiple regressions were used to examine whether sexual harassment victimization and satisfaction with peer -relationships, along with gender and age, could predict levels of depression and anxiety symptoms in our sample. Age made no significant contribution to the model and was, therefore, not included in the final analyses. Intercorrelation analysis between the dependent variables used in the multiple regression was run and was found strong and in the expected direction, r ¼ 0.80; p < 0.01. However, both dependent variables were included due to previous research findings, discussed in the Introduction. To test whether the quality of peer relationships impacted the correlation between dependent and independent variables, interaction analysis was run but found not significant. Swedish version of Beck Youth Inventories of Social and Emotional Impairment (BYI). The BYI is an instrument devel- oped for assessing depression, anxiety, anger, disruptive behaviour, and self-concepts in children. Each sub-scale con- sists of 20 questions, each ranked on a four-grade Likert scale ranging from never (0) to always (3) [19]. Only the depression subscale was used (BYI-D) in the present study. All sub-scales (including BYI-D) have previously shown satis- fying internal consistency with Cronbach’s alpha above 0.80 in a Swedish normative sample [20]. Internal consistency was excellent with a Cronbachs alpha coefficient of 0.94 (CI 95% [0.93 0.95]) in the present study. BYI has been found to be a valid instrument in non-clinical samples [20] in comparison with other instruments that measure similar symptoms. Study setting Data was collected from a survey conducted by the project Adolescents’- experience of mental illness- Psychometric properties of new Swedish versions of tests (UPOP). The S. STÅHL AND I. DENNHAG 332 Statistical analyses Gender See Table 3 for results. Multiple regression analyses showed that the independent variables explained more variance for depressive symptoms (Adj. R2 for girls 0.28; F (4,362) ¼ 36.90, for boys 0.21; F (4,222) ¼ 16.00) than for anxiety symptoms (Adj. R2 for girls 0.20; F (4,362) ¼ 23.34, for boys 0.14; F (4,222) ¼ 10.11). Furthermore, online sexual harass- ment correlated positively with symptoms of mental illness for girls but was not significant for boys. Offline sexual har- assment alone had a stronger correlation with poor mental health on boys than online and offline harassment com- bined. To further analyse the effect of gender on the out- come, an interaction analysis between gender and harassment was run but found not significant. Results from this study confirm results from previous stud- ies that show girls to be more frequently victimized by sex- ual harassment [3–6,24]. A nation-wide survey in Sweden shows that this pattern lingers in adulthood, with more women than men reporting sexual harassment [25]. To our surprise, age showed no significant association with either of the dependent variables. Anxiety symptoms Furthermore, victimization offline alone correlated more than both online harassment and online and offline victimization combined. Prevalence of online and offline sexual harassment Results from this community sample study confirms that sex- ual harassment is a common nuisance among Swedish ado- lescents, especially girls. Despite the widespread use of internet among adolescents, offline remained the most com- mon place for victimization of both girls and boys in the pre- sent study. Although the internet potentially allows anyone to be harassed by anyone, it also enables relatively simple ways to protect oneself, especially from unknown people, for example, by blocking incoming calls or certain contacts on social media. The same possibilities for protection offline are difficult to achieve when the perpetrator, for example, is a classmate. However, teaching children about the risks of the internet is nonetheless important as internet use will likely remain an integrated part of adolescent lifestyle in the fore- seeable future. Depressive symptoms Results from multiple regression analysis showed that gen- der, harassment variables, and peer -relationships also signifi- cantly correlated with participants’ rating of depressive symptoms, see Table 2. The results for depressive symptoms were similar to those for anxiety symptoms, although online and offline harassment combined showed the strongest con- nection to depressive symptoms. In total, 29% of variance in depressive symptom was explained by the model (Adj. R2 ¼ 0.29; F (5,588) ¼ 49.34). We found that gender significantly correlated with higher BYI-D -score and, therefore, performed separate analyses for girls and boys. relationships and harassment variables were run but found not significant. Anxiety symptoms Results from multiple regression analysis showed that harass- ment variables were positively- and satisfaction with peer- relationships along with gender was negatively correlated NORDIC JOURNAL OF PSYCHIATRY 333 with participants’ rating of anxiety symptoms, see Table 2. The independent variables explained 28% of variance in anx- iety symptoms (Adj.R2 ¼ 0.28; F(5,588) ¼ 45.97). Gender had a significant and relatively large correlation with anxiety symptoms, and therefore, we performed analyses separated by gender. Furthermore, victimization offline alone correlated more than both online harassment and online and offline victimization combined. Depressive symptoms Table 1. Prevalence of sexual harassment among boys and girls, Chi2-test and total prevalence of sexual harassment. Total (n ¼ 594) Girls (n ¼ 367) Boys (n ¼ 227) n % n % n % v2 p Phi Online 47 7.91 34 9.26 13 5.73 1.95 0.16 –0.06 Offline 145 24.41 98 26.70 47 20.70 2.42 0.12 –0.07 Online and Offline 50 8.42 46 12.53 4 1.76 19.74 <0.001 –0.19 Not harassed 352 59.26 189 51.50 163 71.81 23.12 <0.001 0.20 Table 2. Results from multiple regression for the total sample. Anxiety (RCADS) Depression (BYI-D) Adj.R2 ß Adj.R2 ß Model 0.28 0.29 Gender –0.29 –0.15 Online 0.11 0.12 Offline 0.20 0.20 Online and Offline 0.18 0.22 Satisfaction with peer-relationships –0.30 –0.40 p < 0.05, p < 0.01, p < 0.001. harassment among boys and girls, Chi2-test and total prevalence of sexual harassment. Table 2. Results from multiple regression for the total sample. Anxiety (RCADS) Depression (BYI-D) Adj.R2 ß Adj.R2 ß Model 0.28 0.29 Gender –0.29 –0.15 Online 0.11 0.12 Offline 0.20 0.20 Online and Offline 0.18 0.22 Satisfaction with peer-relationships –0.30 –0.40 p < 0.05, p < 0.01, p < 0.001. Table 2. Results from multiple regression for the total sample. with participants’ rating of anxiety symptoms, see Table 2. The independent variables explained 28% of variance in anx- iety symptoms (Adj.R2 ¼ 0.28; F(5,588) ¼ 45.97). Gender had a significant and relatively large correlation with anxiety symptoms, and therefore, we performed analyses separated by gender. Furthermore, victimization offline alone correlated more than both online harassment and online and offline victimization combined. with participants’ rating of anxiety symptoms, see Table 2. The independent variables explained 28% of variance in anx- iety symptoms (Adj.R2 ¼ 0.28; F(5,588) ¼ 45.97). Gender had a significant and relatively large correlation with anxiety symptoms, and therefore, we performed analyses separated by gender. Anxiety and depression linked to sexual harassment in girls and boys Gender significantly correlated with anxiety and depressive symptoms. Analyses of girls and boys separately showed that online harassment was significant for girls but not for boys, while at the same time no significant difference in the preva- lence of online harassment was found. Previous studies have found that girls experience multiple types of harassment more frequently than boys, both online [3,12], and offline [26,27]. This was not captured in our study since the number of incidents of sexual harassment were not recorded, and therefore, all participants were weighted equally, irrespec- tively of the amount of harassment they had experienced. The association with mental health from offline sexual har- assment was almost the same for both girls and boys. So, although offline harassment is more commonly targeted towards girls, the connection to mental health, when it occurs, is similar for both girls and boys. Methodological aspects and limitations No claim of causality can be made due to the cross-sectional design used in this study. Although the connection between harassment and poorer mental health has been established through both cross-sectional and longitudinal data, studies have also found the relationship to be reciprocal [9,21,29]. This suggests that adolescents with mental health problems could also be more vulnerable to harassment than others [9]. inclusive environments for adolescents in preventing men- tal illness. inclusive environments for adolescents in preventing men- tal illness. include. Consequently, we hoped to come as close as pos- sible to the real prevalence number. However, the possibility of a response bias cannot be ruled out since participation in the study was optional, and the schools were not ran- domly selected. Clinical relevance Since this study points to the fact that sexual harassment correlates with symptoms of anxiety and depression in a convenience school sample, further studies related to a clin- ical population would be of interest. In the future, it is important for clinical work to know more about the ways in which boys and girls are exposed to sexual abuse online and how they engage in sexual risk behaviours online and offline. Moreover, increased knowledge about online sexual victim- ization is needed for victims, caregivers, as well as for care providers. The knowledge can be used in the assessment of mental illness in clinics, and as a knowledge base in trauma therapy. In clinics, there is a need to ask more about online environments, otherwise teens might not feel that the pro- fessionals can understand them. Satisfaction with peer-relationships As stated in the Introduction, the prevalence numbers of sexual harassment vary within large spans in earlier research. Other studies in Nordic and western countries have found similar or higher prevalence numbers [3,12,24,26], and in most of them, girls were found to be more frequently victi- mized both online and offline. The present study applied an inclusive strategy that used open questions and provided examples of situations that could count as sexual harassment but left to the respondent to decide what situations to Satisfaction with peer-relationships showed the strongest connection to the outcome in all analyses (see Tables 2 and 3). Participants who rated the quality of their peer-relation- ships higher had significantly fewer symptoms of anxiety and depression. The effect was especially large for depressive symptoms, compared to anxiety, and for girls compared to boys. To test our hypothesis that peer-relationship satisfac- tion could significantly influence the effect sexual harass- ment had on mental health, interaction analyses for peer- 334 S. STÅHL AND I. DENNHAG Table 3. Results from multiple regression for girls and boys separately. Anxiety (RCADS) Depression (BYI-D) Girls Boys Girls Boys Adj.R2 ß Adj.R2 ß Adj.R2 ß Adj.R2 ß Model 0.20 0.14 0.28 0.21 Online 0.14 0.06 0.15 0.06 Offline 0.22 0.21 0.20 0.21 Online and Offline 0.21 0.14 0.25 0.16 Satisfaction with peer-relationships –0.34 –0.28 –0.43 –0.37 p < 0.05, p < 0.01, p < 0.001. Table 3. Results from multiple regression for girls and boys separately. inclusive environments for adolescents in preventing men- tal illness. References [1] European Union Agency for Fundamental Rights. Violence against women: an EU-wide survey. Results at a glance. Vienna: European Union Agency for Fundamental Rights; 2014. [18] Barbara Hoff E, Mikael Julius S, Clara T, et al. Assessing the Revised Child Anxiety and Depression Scale (RCADS) in a national sample of Danish youth aged 8–16 years. PLoS One. 2012;7(5): e37339. [2] Swedish Media Council. Ungar & medier 2019. Stockholm: Statens medieråd; 2019. [2] Swedish Media Council. Ungar & medier 2019. Stockholm: Statens medieråd; 2019. [19] Beck JS, A BT, J JB. Manual for the beck youth inventories of emotional and social impairment. San Antonio (TX): The Psychological Corporation; 2001. [3] Zetterstrom Dahlqvist H, Gillander Gadin K. Online sexual victim- ization in youth: predictors and cross-sectional associations with depressive symptoms. Eur J Public Health. 2018;28(6):1018–1023. [19] [20] [3] Zetterstrom Dahlqvist H, Gillander Gadin K. Online sexual victim- ization in youth: predictors and cross-sectional associations with depressive symptoms. Eur J Public Health. 2018;28(6):1018–1023. [20] Beck JS, Beck AT, Jolly JB, et al. Beck ungdomsskalor: manual: svensk version: [bed€omning av emotionell och social problematik hos barn och ungdom]. Psykologif€orlaget; 2017. [4] Kaltiala-Heino R, Fr€ojd S, Marttunen M. Sexual harassment and emotional and behavioural symptoms in adolescence: stronger associations among boys than girls. Soc Psychiatry Psychiatr Epidemiol. 2016;51(8):1193–1201. [21] Zetterstr€om Dahlqvist H, Landstedt E, Gillander Gådin K. Depressive symptoms and the associations with individual, psy- chosocial, and structural determinants in Swedish adolescents. Health. 2012;04(10):881–889. [5] Bendixen M, Daveronis J, Kennair LEO. The effects of non-physical peer sexual harassment on high school students’ psychological well-being in Norway: consistent and stable findings across stud- ies. Int J Public Health. 2018;63(1):3–11. [22] [22] Cohen J. Statistical power analysis for the behavioral sciences. Hillsdale: L. Erlbaum Associates; 1988. [6] Bucchianeri MM, Eisenberg ME, Wall MM, Piran N, et al. 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Satisfaction with peer-relationships Results from the present study underlines the importance of friends as buffers to the mental illness of adolescents. Participants who reported greater satisfaction and felt them- selves to be someone others wanted as a friend reported sig- nificantly lower symptoms of both depression and anxiety. Whether this is mediated because teens who have many friends are possibly victims to a lesser extent or, as has been found before [24,28], the acts of harassment are less distress- ing for victims who have better peer -support, is beyond the scope of this article. Surprisingly and contrary to our hypoth- esis, no significant interaction effect was seen between peer- relationships and sexual harassment. Possibly, the positive effect rendered by good peer-relationships is general and not specifically seen when an adolescent is subjected to sex- ual harassment. The correlation between satisfaction with peer-relationship and less symptoms of mental illness, how- ever, was large for both anxiety and depressive symptoms and for both genders, thus supporting strategies for creating All variables significantly correlated with the outcome for the total sample. However, due to small sample sizes and large confidence intervals in the groups of boys who reported online only (n ¼ 13), and online and offline (n ¼ 4) harassment, no strong conclusions can be drawn from these groups. However, strong significant relationships were found even though the gender groups were small. Participants were not geographically stratified and did not fully match the Swedish pediatric population, for example, the unbalanced gender ratio limited generalizability. As men- tal health is associated with socioeconomics, another limita- tion was a quantified measure of socioeconomics. The instruments used to measure online- and offline sex- ual harassment were not originally meant for this purpose and therefore not ideally designed. Even though some response alternatives might have appeared confusing, the questions were designed to include as many experiences as possible. NORDIC JOURNAL OF PSYCHIATRY 335 Simon Ståhl http://orcid.org/0000-0003-3050-8879 Inga Dennhag http://orcid.org/0000-0001-7336-1657 Simon Ståhl http://orcid.org/0000-0003-3050-8879 Inga Dennhag http://orcid.org/0000-0001-7336-1657 [16] Chorpita BF, Yim L, Moffitt C, et al. Assessment of symptoms of DSM-IV anxiety and depression in children: a revised child anxiety and depression scale. Behav Res Ther. 2000;38(8):835–855. [17] Piqueras JA, Martin-Vivar M, Sandin B, et al. The revised child anxiety and depression scale: a systematic review and reliability generalization meta-analysis. J Affect Disord. 2017;218:153–169. Funding The present research was financially supported by V€asterbotten county council, award number RV-931721, and Swedish Psychiatry fund. [14] Ybarra ML, Leaf PJ, Diener-West M. Sex differences in youth- reported depressive symptomatology and unwanted internet sex- ual solicitation. J Med Internet Res. 2004;6(1):e5. [15] Dooley B, Fitzgerald A, Giollabhui NM. The risk and protective factors associated with depression and anxiety in a national sam- ple of Irish adolescents. Ir J Psychol Med. 2015;32(1):93–105. Mar Disclosure statement [12] Helweg-Larsen K, Sch€utt N, Larsen HB. Predictors and protective factors for adolescent Internet victimization: results from a 2008 nationwide Danish youth survey. Acta Paediatr. 2012;101(5): 533–539. Disclosure statement No potential conflict of interest was reported by the author(s). References [ [25] Sexuell och reproduktion h€alsa och r€attigheter (SRHR) i Sverige 2017 (Public Health Agency of Sweden) 2019. [8] Chiodo D, Wolfe DA, Crooks C, et al. Impact of sexual harassment victimization by peers on subsequent adolescent victimization and adjustment: a longitudinal study. J Adolesc Health. 2009; 45(3):246–252. [26] Montiel I, Carbonell E, Pereda N. Multiple online victimization of Spanish adolescents: Results from a community sample. Child Abuse Negl. 2016;52:123–134. [27] Kaltiala-Heino R, Fr€ojd S, Marttunen M. Sexual harassment victim- ization in adolescence: associations with family background. Child Abuse Negl. 2016;56:11–19. [9] Dahlqvist HZ, Landstedt E, Young R, et al. Dimensions of peer sexual harassment victimization and depressive symptoms in adolescence: a longitudinal cross-lagged study in a swedish sam- ple. J Youth Adolesc. 2016;45(5):858–873. [28] [28] Perez-Gonzalez A, Guilera G, Pereda N, et al. Protective factors promoting resilience in the relation between child sexual victim- ization and internalizing and externalizing symptoms. Child Abuse Neglect. 2017;72:393–403. 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https://openalex.org/W2968331369	https://scindeks-clanci.ceon.rs/data/pdf/0350-0373/2019/0350-03731901077B.pdf	English	null	Financial development and growth: Evidence from Serbia	Industrija	2,019	cc-by-sa	4,616	Industrija, Vol.47, No.1, 2019 1 University of Belgrade, Faculty of Economics, milosbozovic@ekof.bg.ac.rs Financial Development and Growth: evidence from Serbia Article history: Received: 24 January 2019 Sent for revision: 4 February 2019 Received in revised form: 18 March 2019 Accepted: 19 March 2019 Available online: 1 April 2019 Abstract: This paper studies the relationship between financial development and economic growth in Serbia. We focus on the influence of stock market and banking-sector development on growth. Using the framework of a neoclassical growth model, we investigate the impact of stock market liquidity and credit activity of banks on per-capita GDP growth rate. In lines with many previous results in the literature, we find a positive and statistically significant impact of stock market liquidity and bank credit on economic growth. We control for the usual determinants of growth, such as government consumption, foreign direct investments and inflation. Keywords: economic growth, stock market, banks, transition economies Božović1 JEL:G10, G21, O16 DOI:10.5937/industrija47-21454 UDC: 336.1/.2(497.11) 336.71 338.76 Original Scientific Paper Financial Development and Growth: evidence from Serbia JEL:G10, G21, O16 DOI:10.5937/industrija47-21454 UDC: 336.1/.2(497.11) 336.71 338.76 Original Scientific Paper Miloš Božović1 I d t ij V l 47 N 1 2019 1 University of Belgrade, Faculty of Economics, milosbozovic@ekof.bg.ac.rs Finansijska razvijenost i privredni rast: rezultati za Srbiju Apstrakt: Rad se bavi odnosom između finansijske razvijenosti i privrednog rasta u Srbiji. Istraživanje je usredsređeno na uticaj razvoja tržišta hartija od vrednosti i bankarskog sektora na rast. Koristeći okvir neoklasičnog modela rasta, istražujemo uticaj likvidnosti berze i kreditne aktivnosti banaka na stopu rasta bruto domaćeg proizvoda po glavi stanovnika. U skladu sa mnogim prethodnim rezultatima u literaturi, nalazimo pozitivan i statistički značajan uticaj likvidnosti berze i kreditne aktivnosti banaka na ekonomski rast. U Industrija, Vol.47, No.1, 2019 77 Božović M.: Financial Development and Growth: evidence from Serbia regresijama smo koristili kontrole za uobičajene determinante rasta, kao što su državna potrošnja, strane direktne investicije i stopa inflacije. Ključne reči: ekonomski rast, berza, banke, privrede u tranziciji regresijama smo koristili kontrole za uobičajene determinante rasta, kao što su državna potrošnja, strane direktne investicije i stopa inflacije. Ključne reči: ekonomski rast, berza, banke, privrede u tranziciji 1. Introduction Economic growth is often associated with the quality of financial system. Intuitively, a developed financial market and a stable network of financial intermediaries foster more efficient resource allocation and flow of information. Many theoretical models provide predictions that confirm this simple intuition. These models mostly rely on endogenous growth and different roles played by financial system. Levine (2005) presents a detailed review of theoretical and empirical research related do the link between development of capital markets and macroeconomic performance. Empirical evidence shows that a positive influence of financial development on growth typically follows from a decrease in cost of capital in emerging and transition economies, or from an increase in total factor productivity in developed economies (See, for example, Papaioannou, 2007). However, establishing causal relationship from financial development to economic growth empirically has been the crucial issue so far. King and Levine (1993) used a regression across different countries in one of the first attempts to test the existence of such a causality. They found that the size of the country’s banking system relative to its Gross Domestic Product (GDP) predicts long-term growth rates, even when one controls for other explanatory variables. Other measures of financial depth have been used as well. For instance, Levine and Zervos (1998) show a similar causal link between stock market capitalisation and growth. Using instrumental variable approach and introducing controls for other determinants of growth did not change the original results qualitatively (see, for instance, Levine et al., 2000; or Beck and Levine, 2004). Various mechanisms through which financial markets impact economic growth have also been studied. Again, Levine (2005) summarizes the main channels. These predominantly include informational efficiency, better corporate controls and monitoring of projects, efficient allocation of capital, pooling of savings, risk management and diversification, or more efficient exchange of goods and services (Claessens and Laeven, 2003; Demirgüç- Industrija, Vol.47, No.1, 2019 78 Božović M.: Financial Development and Growth: evidence from Serbia Kunt et al., 2006; Beck, 2012). The availability of external finance is positively related to entrepreneurship, innovation and market entry (Klapper et al., 2006; Aghion et al., 2007; Ayyagari et al., 2011). It also facilitates growth and investment opportunities of existing companies (Rajan and Zingales, 1998; Beck et al., 2005, 2006). Small and medium-sized enterprises seem to exhibit a stronger impact of financial depth on firm performance and growth than the large ones (Beck et al., 2005, 2008a, 2008b). 1. Introduction On the flip side, the mechanism that makes finance beneficial to growth also leads to fragility and systemic risk. Although it is one of the core positive roles that financial industry has on the rest of the economy, the maturity and liquidity transformation comes with a side effect, as the system becomes susceptible to shocks, and prone to bank runs and liquidity shortages. The information asymmetries between lenders and borrowers alleviated by the banks is another potential source of fragility due to the agency conflicts between savers and banks. Carletti (2008) provides an overview of different sources of financial fragility. For instance, agency problems also promote increased risk-taking behaviour of banks. The negative externalities of bank failures have made the financial sector among the most regulated ones. They caused the establishment of various safety nets that act as (explicit or implicit) subsidies. This, in turn, incentivises aggressive risk-taking behaviour by the banks, as well as the associated high leverage (Demirgüç-Kunt and Kane, 2002). More recent empirical findings indicate that the conventional finance-growth nexus changed after 1990s, differing across regions, countries, and income levels (Nili and Rastad 2007; Barajas et al., 2013), or firm size (Rioja and Valev, 2004a, 2004b). There might be even some evidence for the disappearance of the link, mostly as a results of more prevalent banking crises than in the earlier data (Rousseau and Wachtel 2011). Some papers also report the so-called “too much finance” effect, when financial development increases growth, but only to the extent beyond which additional deepening reduces it (Arcand et al., 2012; Beck et al., 2012; Aizenman et al., 2015). Possible explanations of this phenomenon may be sought in the negative impact on allocative efficiency and on crowding out of human capital away from more productive sectors of the economy during rapid expansions of financial sector (Cecchetti and Kharroubi, 2015; Dabla-Norris et al., 2015). Rajan (2005) highlights potential “catastrophic meltdowns” in large and complicated financial systems. Gennaioli et al. (2012) show that financial 79 Industrija, Vol.47, No.1, 2019 Božović M.: Financial Development and Growth: evidence from Serbia fragility and systemic risk can be increased by financial innovation even in the absence of leverage if tail risks are neglected. Evidence from emerging markets shows many benefits in terms of growth and stability that follow from financial development. 1. Introduction However, these benefits wane as financial markets and institutions become more developed: they mostly affect the total factor productivity growth, and somewhat less the capital accumulation. The speed of financial development and the extent of regulation are also important for the trade-off between stability and growth in emerging economies. See Sahay et al. (2015) for a detailed study of the relationship between financial development and growth in emerging markets. In this paper, we study the relationship between quantifiable metrics of financial development and economic growth in Serbia. By combining quarterly and monthly data, we investigate the impact of stock market development and credit activity of banks on per-capita GDP growth rate. We control for the usual determinants of growth, such as government expenditure, foreign direct investments and inflation. We find a significant positive influence of stock market liquidity and credit activity of the banking sector on economic growth. To the best of our knowledge, this is the first study of the finance-growth nexus conducted on Serbian data. The remainder of this paper is organized as follows. In Section 2, we describe the methodology used in this research. Section 3 describes the data and the variables. Section 4 presents and analyses the regression results. Section 5 concludes. 2. Methodology Growth accounting is a useful tool for identification of determinants of economic growth of a country (Barro and Sala-i-Martin, 2004). These factors may include natural resources, geography, institutional quality, government policies and many others. In the context of this paper, we will focus on the role of financial institutions. We start from the neoclassical aggregate production function in period 𝑡: Industrija, Vol.47, No.1, 2019 80 Božović M.: Financial Development and Growth: evidence from Serbia 𝑌𝑡= 𝐴𝑡𝐾𝑡 𝛼(𝐿𝑡ℎ𝑡)𝛽, (1) (1) (1) 𝑌𝑡= 𝐴𝑡𝐾𝑡 𝛼(𝐿𝑡ℎ𝑡)𝛽, where 𝑌𝑡 is the output, 𝐴𝑡 is the (Hicks-neutral) level of technology, 𝐾𝑡 is the capital stock, 𝐿𝑡 is the labour force, ℎ𝑡 is the average human capital of workers, while 𝛼 and 𝛽 measure the share of capital and quality-adjusted labour force in the economy, respectively. Under the assumption of non- increasing returns to scale, 𝛼+ 𝛽≤1. Dividing both sides of equation (1) by 𝐿𝑡, we obtain the production function in the intensive form: 𝑦𝑡= 𝐴𝑡𝑘𝑡 𝛼ℎ𝑡 𝛽, (2) (2) 𝑦𝑡= 𝐴𝑡𝑘𝑡 𝛼ℎ𝑡 𝛽, where 𝑘𝑡 is the capital per worker. By taking logs of equation (2) and differentiating over time, we get: 𝑦𝑡̇ 𝑦𝑡= 𝛼 𝑘𝑡̇ 𝑘𝑡+ 𝛽 ℎ𝑡̇ ℎ𝑡+ 𝐴𝑡̇ 𝐴𝑡, (3) 𝑦𝑡̇ 𝑦𝑡= 𝛼 𝑘𝑡̇ 𝑘𝑡+ 𝛽 ℎ𝑡̇ ℎ𝑡+ 𝐴𝑡̇ 𝐴𝑡, (3) (3) where dots indicate time derivatives. Equation (3) relates per-capita output growth to the rate of change of physical capital (usually associated with investments), the rate of change of human capital (usually associated with education) and total factor productivity (i.e. the measure of efficiency of capital and labour utilisation in the production). The last term measures technical change as a Solow residual and includes all factors other than physical- and human-capital investment. The first two terms are typically endogenous, which makes equation (3) a useful analytical device for decomposition of growth factors, rather than a “deeper” structural equation that relates growth to its fundamentals. Most empirical analyses of the effects of financial development on growth apply a variant of the following cross-country dynamic panel regression: ∆ln 𝑦𝑖,𝑡= 𝑎+ 𝑏ln 𝑦𝑖,𝑡−1 + 𝑐′𝑋𝑖,𝑡+ 𝜂𝑖+ 𝜀𝑖,𝑡, (4) (4) ∆ln 𝑦𝑖,𝑡= 𝑎+ 𝑏ln 𝑦𝑖,𝑡−1 + 𝑐′𝑋𝑖,𝑡+ 𝜂𝑖+ 𝜀𝑖,𝑡, ∆ln 𝑦𝑖,𝑡= 𝑎+ 𝑏ln 𝑦𝑖,𝑡−1 + 𝑐′𝑋𝑖,𝑡+ 𝜂𝑖+ 𝜀𝑖,𝑡, (4) where 𝑦 is the logarithm of real per-capita GDP, 𝑋 is the set of explanatory factors, 𝜂 accounts for unobserved country-specific effects and 𝜀 is the error term. 2. Methodology The subscript 𝑖 in equation (4) represents a country, while time-specific Industrija, Vol.47, No.1, 2019 81 Božović M.: Financial Development and Growth: evidence from Serbia effects can be captured through time dummies. (See, for example, Beck and Levine, 2004, and the references cited therein.) The lag of log per-capita GDP, ln 𝑦𝑖,𝑡−1, accounts for the usual convergence term in neoclassical models (Barro and Sala-i-Martin, 2004). Most empirical studies find a significant and negative coefficient associated with this convergence term, if one controls for other growth factors 𝑋. This indicates that if a country is further away from the steady state it will have a higher GDP growth rate. The set of growth factors 𝑋 typically includes a proxy for accumulation of human capital (e.g. changes in years of schooling or student enrolment rates) and other controls that may affect productivity, including a proxy measure of financial development. In this paper, we analyse the impact of financial development on growth by running a simpler version of the regression given by equation (4), applied on a single country. More specifically, we estimate a time-series regression of the form: ∆ln 𝑦𝑡= 𝑎+ 𝑏ln 𝑦𝑡−1 + 𝑐′𝑋𝑡+ 𝜀𝑡. (5) (5) ∆ln 𝑦𝑡= 𝑎+ 𝑏ln 𝑦𝑡−1 + 𝑐′𝑋𝑡+ 𝜀𝑡. We use the following set of controls appropriate for a single-country regression:  government expenditure (relative to GDP);  foreign direct investments (relative to GDP);  inflation rate;  a measure of stock market development;  a measure of banking sector development. To capture the stock market development, we follow Beck and Levine (2004) and use turnover ratio as a measure of market liquidity, calculated as the value of traded shares divided by the market value of listed shares. To capture the banking sector development, we follow Rousseau and Wachtel (2000) and use M3 divided by GDP. Intuitively, the expansion of M3 monetary aggregate should serve as a proxy for credit activity of the banking sector. An alternative indicator, calculated as the value of commercial bank claims on the private sector divided by GDP, suggested by Levine and Zervos (1998) would certainly be a better one, as argued by Beck and Levine (2004). However, it was not possible to use such measure in this study due to relatively short time span of the available data on bank claims. 2. Methodology For similar reasons, we also do not 82 Industrija, Vol.47, No.1, 2019 Božović M.: Financial Development and Growth: evidence from Serbia include a proxy for human capital accumulation, although it would be a reasonable control variable. Annual data on primary- and secondary-school enrollment rates are available from UNESCO Institute for Statistics database. For Serbia, they cover the period between 1999 and 2017. Annual data on government expenditures on education as a fraction of GDP (suggested by Barro and Sala-i-Martin, 2004 as a proxy for educational quality) are available from the same source, for the period between 2007 and 2016, with a gap in 2013. 3. Data Our data consist of the real per-capita GDP growth rate as the dependent variable, and the following explanatory variables:  lagged GDP per capita;  government consumption as a fraction of GDP;  foreign direct investments (FDI) as a fraction of GDP;  inflation rate;  turnover ratio;  relative broad money (M3) as a fraction of GDP. The dataset is organised in monthly time series and covers the period between January 2002 and January 2019. Quarterly data on GDP were obtained from the statistical database of the National Bank of Serbia (www.nbs.rs). They are available in constant and current prices in Serbian Dinars (RSD). Data on Serbian population for each year between 2002 and 2018 were taken from the World Bank (data.worldbank.org). Quarterly series of per-capita GDP was then constructed for each quarter by dividing GDP level by the number of inhabitants in the respective year. Monthly data on real GDP per capita were obtained by interpolating the quarterly levels with the growth rate of index of industrial production, available from the database of Statistical Office of the Republic of Serbia with a monthly frequency. Inflation rate was calculated from the consumer price index (CPI), by combining databases of the Statistical Office of the Republic of Serbia and the National Bank of Serbia. Government consumption as a fraction of GDP is available from the World Bank database (we use the same value for each month in a quarter). Data on net FDI in Euros are available from three separate reports of the National Bank of Serbia. They are then recalculated in RSD using the data 83 Industrija, Vol.47, No.1, 2019 Božović M.: Financial Development and Growth: evidence from Serbia on exchange rates available from the same source. Data on market capitalisation and turnover are available from the Belgrade Stock Exchange. For the sake of consistency, we use only data related to stocks, excluding other securities traded on the Exchange. Monetary aggregates are available with monthly frequency from the database of the National Bank of Serbia. Summary statistics for the key explanatory variables – the indicators of financial development – are shown in Table 1. Table 1. 3. Data Summary statistics for the indicators of financial development (monthly data) Statistic Turnover ratio M3/GDP Number of observations 205 205 Mean 0.0220 0.3116 Standard deviation 0.0495 0.1722 Min 0.0005 0.0405 Median 0.0045 0.3406 Max 0.1980 0.6863 Sources: National Bank of Serbia; The World Bank; Statistical Office of the Republic of Serbia; Belgrade Stock Exchange. Table 1. Summary statistics for the indicators of financial development (monthly data) Sources: National Bank of Serbia; The World Bank; Statistical Office of the Republic of Serbia; Belgrade Stock Exchange. 4. Results and Discussion The regression results are summarised in Table 2. We use an ordinary least- squares estimator with robust standard errors and monthly seasonal dummies. Seasonality is driven by the industrial production index, which is used for interpolation of quarterly GDP data. Four factors turn out to be highly significant: lagged GDP per capita, government consumption to GDP, growth rate of turnover ratio and logarithm of M3 to GDP. Together with nine out of eleven seasonal dummies, they explain around 85 percent of the variations in GDP growth rate. The F-statistic is also above the critical value for this regression (3.37 at 0.05 significance level). The sign of the coefficient corresponding to initial GDP per capita is negative, which is in lines with basic economic intuition related to convergence. Foreign direct investments do not influence growth significantly. This in accord with the results of Beck and Levine (2004) obtained on a panel of 40 countries. However, in contrast to Beck and Levine (2004), we find that government consumption has a significant but negative impact on economic growth. The influence of government expenditure on growth is, in general, unclear, as the Industrija, Vol.47, No.1, 2019 84 Božović M.: Financial Development and Growth: evidence from Serbia empirical evidence is inconclusive. Our result is consistent with Butkiewicz and Yanikkaya (2011) although Alexiou (2009) provides the opposite evidence for South-Eastern Europe. An interesting view on this controversy, including the emphasis on the role of interaction of government consumption and some other explanatory variables, is given by d’Agostino et al. (2016). Inflation rate is not significant at 0.05 level, again in lines with Beck and Levine (2004). The point estimate has the expected negative sign, indicating a dampening effect of inflation on GDP growth. Table 2. Regression results (seasonal dummies excluded) Table 2. Regression results (seasonal dummies excluded) Regressors Coefficient Constant 4.6656* (0.8551) Lagged GDP per capita† –0.3221* (0.0578) Government consumption/GDP –0.0094*** (0.0031) FDI/GDP –0.0491 (0.1542) Inflation rate (CPI) –0.0014* (0.0007) Turnover ratio (log diff.) 0.0194* (0.0057) M3/GDP† 0.0414* (0.0104) R2 0.8545) F-statistic 56.36**) Standard errors are reported in parentheses. – p-value < 0.10; – p-value < 0.05; * – p-value < 0.01. † This regressor was included as the logarithm of the variable. Source: Author’s own estimations. Source: Author’s own estimations. Source: Author’s own estimations. Despite the underdevelopment of the stock market, its liquidity is positively correlated with the economic growth. 4. Results and Discussion The expansion of the stock exchange in Serbia was predominantly associated with the transition process, since the shares of government-owned enterprises were sold (directly or indirectly) through the exchange. The sharpest increase in the stock market 85 Industrija, Vol.47, No.1, 2019 Božović M.: Financial Development and Growth: evidence from Serbia capitalisation occurred between 2001 and 2008, after the Property Transformation Law of 2001 was implemented. The largest trading volumes were achieved between 2006 and 2008. This period coincides with one of the largest expansions of the Serbian economy in recent history, with an average growth rate of 6.2% per year. Global Financial Crisis brought a drastic fall in the turnover ratio during 2008 and in the first quarter of 2009. The ratio of M3 to GDP, used as a proxy for credit activity of the banking sector, is significant and positive. Thus, the credit activity of banks seems to have had a major impact on growth. Both empirical and anecdotal evidence suggests that bank credit continues to be the major source of financing in Serbia, at least for large companies (see, for example, OECD 2017). The credit expansion has certainly more direct impact on GDP than the capital raised through the stock exchange. Nevertheless, the existence of a functional, deep and liquid financial market results in many positive externalities for the entire economy and the country’s legal system. Our findings corroborate this intuition. 5. Conclusion In this paper we study the relationship between financial development and economic growth in Serbia. Using a linear regression based on a neoclassical endogenous growth model, we find empirical evidence that the overall financial development is positively associated with economic growth. We reject the hypothesis that the indicators of stock market development and banking activity are unrelated to growth. Convergence factor, measured through the lagged GDP per capita, is also highly significant. We control for the usual growth determinants, such as government consumption, foreign direct investments and inflation. In this paper we study the relationship between financial development and economic growth in Serbia. Using a linear regression based on a neoclassical endogenous growth model, we find empirical evidence that the overall financial development is positively associated with economic growth. We reject the hypothesis that the indicators of stock market development and banking activity are unrelated to growth. Convergence factor, measured through the lagged GDP per capita, is also highly significant. We control for the usual growth determinants, such as government consumption, foreign direct investments and inflation. The results are consistent with theories that emphasise an important positive role of financial markets and institutions on economic growth. They are also very much in lines with the findings summarised by Beck and Levine (2004), Levine (2005), Beck (2012), Sahay et al. (2015) and many other authors. Future research on this topic in Serbia would certainly benefit from an extended dataset, which would span longer periods of time or include additional measures of financial development, particularly on the banking- sector side. Additionally, a more exhaustive study could include other Industrija, Vol.47, No.1, 2019 86 Božović M.: Financial Development and Growth: evidence from Serbia countries that are similar (e.g. transition or post-transition economies, South- Eastern Europe, etc.) Our results imply that creation of an economic and legal environment in which financial markets and institutions can fully develop should be high on the policy agenda. References Aghion, P., Fally, T. and Scarpetta, S. (2007), “Credit Constraints as a Barrier to the Entry and Post Entry Growth of Firms”, Economic Policy Vol. 22, 731–779. Aizenman, J., Jinjarak, Y. and Park, D. 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https://openalex.org/W3172663497	https://journal.kdpu.edu.ua/apd/article/download/1889/1750	Russian	null	Виртуальность как атрибут пространства и времени современного социума	Aktualʹnì problemi duhovnostì	2,017	cc-by	2,748	Актуальнi проблеми духовностi: зб. наук. праць / Ред.: Я.В. Шрамко Вип. 17. — Кривий Рiг, 2016, 219–226 219 Актуальнi проблеми духовностi: В.Н. Сокольчик Социальное пространство и социальное время относятся к важней- шим онтологическим основаниям бытия социума, оформляющим бы- тие современной цивилизации. Параметры социальных пространства и времени во многом определяют образ реальности в сознании совре- менного человека. Тем не менее, социальное пространство и время не совпадают по своим характеристикам с объективно-научным восприя- тием и объяснением этих феноменов. Социальное пространство и вре- мя, во-первых, личностно ориентированы и субъективно наполнены, а, во-вторых, подвержены постоянным динамическим изменениям, со- путствующим и порой предвосхищающим развитие социума. Так, ха- рактеризуя социальное пространство, П. Бурдье в статье «Физическое и социальное пространство» писал о том, что оно является «объекти- вацией и натурализацией прошлых и настоящих социальных отноше- ний» [1, c. 53]. Важнейшей характеристикой социального пространства и социаль- ного времени XXI века, тесно связанной с процессами информатиза- ции и медиатизации общества, является виртуальность. Вне феноме- на виртуальности вообще невозможно понять сущность современной цивилизации, выявить ее характерные особенности, атрибутивные ха- рактеристики и воспринять уклад ее жизни. В контексте развития информационных технологий трансформа- ция термина «виртуальность» изменяется от характеристики состоя- ния реальности (ирреальности) объекта (окружающего нас мира) к 220 Виртуальность как атрибут пространства и времени современному пониманию виртуальности как состояния сознания вос- принимающего субъекта, в котором грань между реальным и скон- струированным мирами фактически стирается. Отчасти в современ- ном дискурсе происходит метафизическое сближение понятий симу- лякр и виртуальность, с той разницей, что симулякр претендует на то, что он обозначает объект, а виртуальность — состояние и процесс. Изучение феномена существования человека в информационном обществе и виртуализацию жизни как следствие информатизации од- ними из первых начали рассматривать представители постмодернизма (Ж. Бодрийяр, Ж. Делез, Ф. Гваттари, У. Эко и др.). Ими осмыслены парадоксы работы с информационными технологиями (например, фе- номен «компьютерного отчуждения»), понятие «виртуальной реаль- ности», симулирующей действительность и превращающей человека в объект манипулирования. Проблемы информационного общества и влияние виртуальности на жизнь социума поднимали и такие извест- ные авторы как Е. Масуда, М. Кастельс, М. Маклюэн, А.В. Гулыга и мн. др. В тоже время виртуальность в аспекте ее влияния и прояв- ления в социальном времени и пространстве остается малоизученной, несмотря на то, что виртуальная реальность прочно вошла в жизнь социума и «проникла» во все его сферы и институты. Феномен устойчивости и даже незыблемости пространства, в кото- ром существует социум, виртуальностью разрушается. Виртуальность привносит в субъективное ощущение пространства текучесть и непо- стоянство, объективируя симуляции и симулякры. При этом реаль- ность становится фантомом, обманом, а ирреальность и симуляция — объективной данностью. В.Н. Сокольчик Применительно к описанию современного со- циума можно даже говорить о формировании особой «вирто-сферы», которая существует в среде «вирто-пространства» и «вирто-времени». Как считает автор теории «виртуального общества» немецкий социо- лог А. Бюль, с развитием технологий виртуальной реальности ком- пьютеры из вычислительных машин превращаются в универсальные машины по производству «зеркальных» миров, пространство которых существует фактически объективно, но формируется по законам новой реальности [8]. Виртуальность как новый атрибут социального пространства, по- рожденный информатизацией общества, активизирует процесс объек- тивации идей, стимулируя моделирование и существование их в про- странстве социума вне материального посредника. Благодаря вирту- альному пространству человек развивает новые виды деятельности: например, он уже не только создает образы, но легко манипулиру- 221 В.Н. Сокольчик В.Н. Сокольчик ет ими, «вживляет» их в ткань повседневности, придает им культур- ный контекст, транслирует любому адресату и т.д. Правда, в резуль- тате возникают не артефакты культуры, а виртуальные «симулякры» (двойники, точные копии действительности, вместе с тем реально не существующие), подчиняющиеся собственной логике и живущие соб- ственной жизнью. Олицетворением виртуальности пространства социума XXI века становится информационная сеть Интернет, гигантская паутина, не только опутывающая пространство, но и реально созидающая новую реальность и формирующая совершенно новую социальную органи- зацию. Сегментом порожденных виртуальностью организаций стано- вятся виртуальные сообщества — новообразования, которые являются по сути и статусу симуляциями или даже симулякрами воображаемой реальности. Примерами могут служить виртуальный бизнес и финан- совое партнерство, виртуальные молитвенные собрания, виртуальные игровые группы и корпорации, виртуальные совместные проекты и даже (!) виртуальный секс и виртуальные кладбища. ( ) у у Отличной от традиционной и общепринятой в виртуальном про- странстве становится система коммуникации индивидов и самовоспри- ятия личности. Коммуникация в виртуальном пространстве вытесня- ется иллюзией, при этом реальность общения заменяется симуляцией последнего. Каждый может «поболтать в чате», через социальные сети связаться с друзьями, знакомыми и незнакомыми, однако в большин- стве случаев в такой коммуникации присутствует не реальная лич- ность, а ее виртуальный образ (аватар), идеализированный сообразно ситуации и не существующий вне виртуального пространства компью- терной реальности. Безусловно, поколение, привычно ощущающее себя свободно в вир- туальной среде, никогда не откажется от всемогущества, предлагаемо- го виртуальным общением. Без мобильности, быстроты и вседозволен- ности, создаваемыми виртуальной средой, современный человек себя уже не представляет. Однако платой за эти реалии является сурро- гатный характер такого вида коммуникации: виртуальное простран- ство не абсолютно тождественно реальному, а виртуальное общение не является адекватной заменой живого непосредственного контакта между людьми, скорее — прообразом зарождающейся коммуникации аватаров. Человек информационного общества, проводящий большую часть жизни в пространстве Интернета, постепенно растворяется в виртуальном пространстве и теряет целостность собственной лично- сти среди симуляций, утрачивая адекватность самовосприятия. 222 Виртуальность как атрибут пространства и времени Коммуницируя с фантомами, сам являясь своего рода фантомом (аватар-маска, реальность которой предполагается, но не гарантиру- ется), человек, живущий в виртуальном пространстве компьютерной сети, начинает путать реальность и сеть, истинное и фантазийное, правду и вымысел. Человек эпохи модерна — эпохи доинформацион- ной, осмысляющий себя в социальной жизни, воспринимает реаль- ность как объективную данность, как естественную необходимость, в которой приходится жить и коррелировать свои действия сообразно внешней довлеющей реальности. В.Н. Сокольчик Человек эпохи постмодерна и инфор- мационного общества, погруженный в виртуальную реальность, увле- ченно «живет» в ней, при этом сознавая ее условность, управляемость параметров, предполагает возможность выхода или, наоборот, «зави- сания» в виртуальном пространстве. Вследствие этого «погружения» перспектива того, что отношения между людьми примут форму от- ношений между образами, становится наиболее реальной перспекти- вой виртуализации общества. Так, в современном обществе симуля- ций происходит переход от стабильного и самобытного (идентичного) «я», где самость воспринимается как нечто постоянное, на изменчи- вое «я», понимаемое как процесс, не имеющий константных точек и позиций. В этом контексте появляется возможность трактовать обще- ственные изменения в пространстве современной цивилизации с по- мощью дихотомии реальное/виртуальное, где взаимодействия между людьми могут осуществляться и реально, и ирреально — как виртуаль- ные аналоги реальных социальных взаимодействий. Смешение реаль- ного и виртуального, возможного и невозможного приводит к тому, что исполнение социальных ролей все чаще заменяется симуляцией, атрибуты институциональности замещаются фантомами, коммуника- тивное пространство стабильности переходит в пространство потоков, сетей и симуляций. По нашему мнению, знаменитое выражение М. Маклюэна, в кото- ром пространство информационного общества трактуется как «гло- бальная деревня» [6, с. 47], уже не отвечает реалиям виртуального про- странства социума XXI века. Краткосрочность общения, неустойчи- вость и кратковременность социальных связей, симуляционный харак- тер отношений, отсутствие устойчивых духовных установок, противо- речивость коммуникативных принципов — эти характеристики более адекватно описываются термином «глобальный мегаполис». Послед- ний является образом и одновременно слепком современной социаль- ной действительности, укорененной в виртуальной среде и выражаю- щей конфликт людей, вынужденно связанных между собой условиями 223 В.Н. Сокольчик образа жизни мегаполиса, но имеющих право на независимую личную жизнь. образа жизни мегаполиса, но имеющих право на независимую личную жизнь. Таким образом, жизнь современного человека протекает сегодня не столько в пространстве объективного мира, сформированного разумом, традициями культуры, биологическими закономерностями, сколько в пространстве фантомном, подчиненном законам виртуаль- ности и симуляциям. Тесно связано с концептуальным определением пространства со- временной цивилизации как виртуального и определение социально- го времени. Последнее неотделимо от пространства, по сути, являясь его проекцией. Рассматривая идею социального времени (социотемпо- ральности) в своих работах известный исследователь проблемы вре- мени Дж. Фрейзер пишет о том, что социальное время воспринимает историческое и психологическое время сквозь призму существования и предпочтений современного общества [9, с. 14]. Таким образом, социо- темпоральность отличается неуниверсальностью времени, его зависи- мостью от людей, восприятий, социотехнологий и др. Интересна идея Фрейзера о том, что социотемпоральность включает в себя разделение на «время ощущаемое и время понимаемое». В.Н. Сокольчик «Время понимаемое» от- носительно объективно и встраивается в пирамиду темпоральностей Фрейзера (см., напр.: [3]), но «время ощущаемое» (по Фрейзеру, оно является порождением научно-промышленной цивилизации и своего рода коллективной шизофренией) абсолютно субъективно не только на личностном, но и на общественном уровне. Специфической характеристикой социального времени современ- ной эпохи является его виртуальность. «Вирто-время» можно сжи- мать и развертывать, направлять вспять (инверсия времени) или же в будущее, работать с ним, нарушая все известные законы объективно- го мира (в целом на этом строятся и компьютерные эксперименты, и компьютерные игры). Виртуальное время, являющееся прерогативой «вирто-сферы», в силу тотальной компьютеризации общества посте- пенно переносится в реальную жизнь социума. К.Ю. Галкин в своем размышлении о виртуальном времени пишет о преимуществах его в аспекте восприятия социума: Реальное время — все старит, снимает покров иллюзорности, вирту- альное время — напротив, создает иллюзию всесильности над вечно- стью и энтропией, обольщая человека надеждой на бессмертие [2]. Порождением виртуального времени становится личность, неадекватно оценивающая окружающее, личность, не стремящаяся к объективности мира-идеала, а живущая в иллюзии реальности фанто- мов. Мир и социум для индивида превращаются в разупорядоченную реальность — события наслаиваются, не уходя в прошлое, сосуществу- ют с нынешними и даже будущими. Процесс этот, безусловно, закономерен и, хотя воздействие вирту- альности на социальное время тревожит психологов, социологов и про- сто думающих людей, вряд ли это можно остановить или предотвра- тить дальнейшее тотальное распространение. Для адаптации личности и социума в целом к современным условиям бытия, в т. ч. к социаль- ному времени и пространству, обществу настоятельно требуются раз- работки новых социальных механизмов стабилизации культурно-ком- муникативных процессов, упорядочения коммуникативного простран- ства, усиление доверия к науке нового века — ее объясняющей и про- гнозирующей функции. Только научившись противостоять деструк- тивному влиянию быстротечности, фрагментарности, иллюзорности и нестабильности социального пространства и времени современной ци- вилизации человечество сможет адекватно воспринять будущее и со- вершенствовать свой жизненный мир. Реальное время — все старит, снимает покров иллюзорности, вирту- альное время — напротив, создает иллюзию всесильности над вечно- стью и энтропией, обольщая человека надеждой на бессмертие [2]. Реальное время — все старит, снимает покров иллюзорности, вирту- альное время — напротив, создает иллюзию всесильности над вечно- стью и энтропией, обольщая человека надеждой на бессмертие [2]. Виртуальное время характеризуется «неуниверсальностью» (спе- цифичностью), т. е. своего рода индивидуальностью; дискретно- 224 Виртуальность как атрибут пространства и времени стью — адекватным сочетанием линейного и циклического понимания хода времени; обратимостью как возможностью исправить ошибки и переиграть сложившееся; беспредельностью как победой над конеч- ностью и, значит, ограниченностью человека и цивилизации в целом; неравномерностью хода, что позволяет социуму вписываться в нераз- меренный, убыстряющийся ритм постиндустриального общества [7, с. 193]. Особенную позитивную окраску виртуально трактуемому вре- мени придает кажущаяся несерьезность, легкость и «вседоступность» (вседозволенность) нового социального времени XXI века. Собственно виртуальность как черта социального времени пост- индустриальной цивилизации, породила и новые отношения между людьми — отношения отчасти виртуальные, а именно временные и ускоренно-облегченные (которые точно отражает сленговое выраже- ние «не заморачиваться»), неукорененные. Еще одна черта социального «вирто-времени» современной инфор- мационной цивилизации — иллюзия свободного распоряжения и управ- ления временем. Виртуальное время подвластно социуму и его «прихо- тям», следовательно, оно не является независимым и объективным, по- стоянным, стабильным и размеренным. Социальная темпоральность, сформированная обществом XXI века, предполагает такой функци- ональный подход к времени, который подчеркивает иллюзорность и субъективность последнего: возможность ускорить время (улучшить динамику процессов), «перевернуть время» (перевод часов), обогнать или затормозить время (перелететь на самолете через несколько часо- вых поясов), структурировать время и соответственно его подчинить (сезонная работа, учеба, долгосрочные планы и пр.). Впрочем, это гло- бальное управление виртуальным временем вопреки внешней иллюзии не создает естественного ритма, подвластного человеку, а вводит в дей- ствие некий функционал, постепенно подавляющий и разрушающий индивида [5, с. 432]. В этом контексте личность становится зависимой от сконструированных социумом виртуальных обстоятельств (вирту- альных циклов, ритмики процессов). Интересно, что те характеристики, которые привносит в восприя- тие времени виртуальность, одинаково необходимы для современной науки с ее дилеммами и парадоксами (виртуальность времени позво- ляет сочетать несочетаемое, реализовать принцип дополнительности и исключить строгий детерминизм, предлагая свободу трактовки собы- тий (фактов), свободу эксперимента, свободу прогнозов) также необ- ходимы и для индивида, поскольку позволяют иллюзиям всемогуще- ства возобладать над реальностью бессилия. В поисках себя в усло- 225 В.Н. Сокольчик виях все большего погружения в реальность «вирто-сферы», человек все меньше ориентируется на «другого», соответственно все меньше видит себя «в зеркале другого» и все дальше уходит от объективно- го мира. М. Кастельс писал: История только начинается, если понимать под ней то, что после тыся- челетий доисторической битвы с природой, сначала выживая в борьбе с ней, а затем покоряя ее, человеческий вид вышел на такой уровень знаний и социальной организации, который дает нам возможность жить в преимущественно общественном мире. Речь идет о начале ино- го бытия, о приходе нового, информационного века, отмеченного са- мостоятельностью культуры по отношению к материальной основе на- шего существования. Но вряд ли это может послужить поводом для большой радости, ибо, оказавшись в нашем мире наедине с самими собой, мы должны будем посмотреть на свое отражение в зеркале ис- торической реальности. То, что мы увидим, вряд ли нам понравится [4, с. 505]. Социальное пространство и социальное время — реальные атрибу- ты существования социума. Их характеристики, свойства, особенности формируют картину мира и образ общества в понимании человека. От- нюдь не объективные научные представления о пространстве и време- ни, а социально сформированные конструкты последних оказывают 226 Виртуальность как атрибут пространства и времени влияние на нашу повседневную жизнь, формируя ее стиль, строй и ритм. Все больше подпадая под порабощение социального обустрой- ства жизни, человек оказывается все дальше от объективности и раз- меренности мира природы и все сложнее различает истину и ложь, реальность и симуляцию, виртуальные и действительные возможно- сти. Анотацiяiяя Сокольчик В.М. ртуаль сть як атрибут простору часу сучас ого соцiумууму к В.М. ртуаль сть як атрибут простору часу сучас ого соцiумууму Стаття присвяче а характеристицiуму соцiумуаль ого часу соцiумуаль ого простoруру сучас ої формацiумуй ої цiумуивлзацiумуї. Автор розглядає ов цiуму ост, характеристики суспльства, кому катив практики вд оси и, що ви икли в аслдок с ува я суспльства у вртуаль ому простор вртуаль ому час. Ключовi словаiя словi словаа: соцiумуаль ий простр, соцiумуаль ий час, вртуаль сть, симулякр, формацiумуй е суспльство. Литература [1] Бурдье П. Физическое и социальное пространства // Социология социального пространства. — СПб. : Алтейя, 2007. [2] Галкин К.Ю. Размышление о виртуальном времени [Электронный ресурс]. — Режим доступа: http://biblio- fond.ru/view.aspx?id=105427♯1. [3] Гансвинд И.Н. Дж.Т. Фрейзер и его теория време- ни [Электронный ресурс]. — Режим доступа: http:// www.chronos.msu.ru/old/RREPORTS/gansvind_o_freizere.htm [4] Кастельс М. Становление общества сетевых структур // Новая постиндустриальная волна на Западе. — М. : Academia, 1999. — C. 494-505. [5] Кастельс М. Информационная эпоха: экономика, общество и культура. — М. : ГУ ВШЭ, 2000. [6] Мак-Люэн М. Галактика Гуттенберга. Сотворение человека пе- чатной культуры. — К. : Ника-Центр, 2003. [7] Сокольчик В.Н. Время, порожденное социумом 21 века, и его вли- яние на человека и общество // Системная трансформация об- щества: инновации и традиции. — Брест, 2013. — Вып. 10. — C. 191- 194. [8] B¨uhl A. Die virtuelle Gesellschaft des 21. Jahrhunderts: sozialer Wandel im digitalen Zeitalter. — Wiesbaden : Westdeutscher Verlag, 2000. [9] Fraser J.T. A Backward and Forward Glance // Study of Time IV. — N.Y., 1981. Надiйшла до редакцiї 10 червня 2016 р. Abstract Sokolchyk V.М. Vrtualty as an attrbute oруf space and tme oруf the moруdern soруcety The paper s devoруted toру a characterzatoруn oруf a soруcal tme and soруcal space oруf the moруder nfoруrmatoруnal cvlzatoруn. The authoруr coруnsders new values, characterzatoруns oруf soруcety, coруmmuncatve practces and relatoруnshps arsng due toру soруcal exstence n a vrtual space and vrtual tme. Keywords: Soруcal space; soруcal tme; vrtualty; smulacrum; nfoруrmatoруn soруcety References 1. Burdje P. Fzcheskoруe soруcalnoруe proруstranstva // Soруcoруloруgya soруcalnoруgoру proруstranstva. — SPb. : Altejya, 2007. 2. Galkn K.YU. Razmyshlene oру vrtualnoруm vremen : http://bbloруfoруnd.ru/vew.aspx?d=105427]1. 3. Gansvnd I.N. Dzh.T. Frejzer egoру teoруrya vremen : http:// www.chroруnoруs.msu.ru/oруld/RREPORTS/gansvnd_oру_frezere.htm 3. Gansvnd I.N. Dzh.T. Frejzer egoру teoруrya vremen : http:// www.chroруnoруs.msu.ru/oруld/RREPORTS/gansvnd_oру_frezere.htm 4. Kastels M. Stanoруvlene oруbshchestva setevyh struktur // Noруvaya poруstndustralnaya voруlna na Zapade. — M. : Academa, 1999. — C. 494-505. 4. Kastels M. Stanoруvlene oруbshchestva setevyh struktur // Noруvaya poруstndustralnaya voруlna na Zapade. — M. : Academa, 1999. — C. 494-505. 5. Kastels M. Infoруrmacoруnnaya ehpoруha: ehkoруnoруmka, oруbshchestvoру kultura. — M. : GU VSHEH, 2000. 5. Kastels M. Infoруrmacoруnnaya ehpoруha: ehkoруnoруmka, oруbshchestvoру kultura. — M. : GU VSHEH, 2000. 6. Mac-Luen M. Galaktka Guttenberga. Soруtvoруrene cheloруveka pechatnoруj kultury. — K. : Nka-Centr, 2003. 7. Soруkoруlchk V.N. Vremya, poруroруzhdennoруe soруcumoруm 21 veka, egoру vlyane na cheloруveka oруbshchestvoру // Sstemnaya transfoруrmacya oруbshchestva: nnoруvac tradc. — Brest, 2013. — Vyp. 10. — C. 191- 194. 8. Bühl A. De vrtuelle Gesellschaft des 21. Jahrhunderts: soруzaler Wandel m dgtalen Zetalter. — Wesbaden : Westdeutscher Verlag, 2000. 9. Fraser J.T. A Backward and Foруrward Glance // Study oруf Tme IV. — N.Y., 1981.
https://openalex.org/W2272209815	https://research.monash.edu/files/226268065/150280405_oa.pdf	English	null	A scoping review of competencies for scientific editors of biomedical journals	BMC medicine	2,016	cc-by	12,122	© 2016 Galipeau et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. RESEARCH ARTICLE Open Access A scoping review of competencies for scientific editors of biomedical journals James Galipeau1* , Virginia Barbour2, Patricia Baskin3,13, Sally Bell-Syer4,14, Kelly Cobey1,15, Miranda Cumpston5,16, Jon Deeks6, Paul Garner7, Harriet MacLehose8, Larissa Shamseer1,10, Sharon Straus9, Peter Tugwell1,10,17, Elizabeth Wager11, Margaret Winker12 and David Moher1,10 * Correspondence: jgalipeau@ohri.ca 1Clinical Epidemiology Program, Ottawa Hospital Research Institute, Ottawa, Canada Full list of author information is available at the end of the article A scoping review of competencies for scientific editors of biomedical journals James Galipeau1* , Virginia Barbour2, Patricia Baskin3,13, Sally Bell-Syer4,14, Kelly Cobey1,15, Miranda Cumpston5,16, Jon Deeks6, Paul Garner7, Harriet MacLehose8, Larissa Shamseer1,10, Sharon Straus9, Peter Tugwell1,10,17, Elizabeth Wager11, Margaret Winker12 and David Moher1,10 James Galipeau1* , Virginia Barbour2, Patricia Baskin3,13, Sally Bell-Syer4,14, Kelly Cobey1,15, Miranda Cumpston5,16, Jon Deeks6, Paul Garner7, Harriet MacLehose8, Larissa Shamseer1,10, Sharon Straus9, Peter Tugwell1,10,17, Elizabeth Wager11, Margaret Winker12 and David Moher1,10 Abstract Background: Biomedical journals are the main route for disseminating the results of health-related research. Despite this, their editors operate largely without formal training or certification. To our knowledge, no body of literature systematically identifying core competencies for scientific editors of biomedical journals exists. Therefore, we aimed to conduct a scoping review to determine what is known on the competency requirements for scientific editors of biomedical journals. Methods: We searched the MEDLINE®, Cochrane Library, Embase®, CINAHL, PsycINFO, and ERIC databases (from inception to November 2014) and conducted a grey literature search for research and non-research articles with competency-related statements (i.e. competencies, knowledge, skills, behaviors, and tasks) pertaining to the role of scientific editors of peer-reviewed health-related journals. We also conducted an environmental scan, searched the results of a previous environmental scan, and searched the websites of existing networks, major biomedical journal publishers, and organizations that offer resources for editors. Results: A total of 225 full-text publications were included, 25 of which were research articles. We extracted a total of 1,566 statements possibly related to core competencies for scientific editors of biomedical journals from these publications. We then collated overlapping or duplicate statements which produced a list of 203 unique statements. Finally, we grouped these statements into seven emergent themes: (1) dealing with authors, (2) dealing with peer reviewers, (3) journal publishing, (4) journal promotion, (5) editing, (6) ethics and integrity, and (7) qualities and characteristics of editors. Discussion: To our knowledge, this scoping review is the first attempt to systematically identify possible competencies of editors. Limitations are that (1) we may not have captured all aspects of a biomedical editor’s work in our searches, (2) removing redundant and overlapping items may have led to the elimination of some nuances between items, (3) restricting to certain databases, and only French and English publications, may have excluded relevant publications, and (4) some statements may not necessarily be competencies. Conclusion: This scoping review is the first step of a program to develop a minimum set of core competencies for scientific editors of biomedical journals which will be followed by a training needs assessment, a Delphi exercise, and a consensus meeting. Keywords: Biomedical, Competencies, Journal, Scientific editor, Scoping review Full list of author information is available at the end of the article © 2016 Galipeau et al. Galipeau et al. BMC Medicine (2016) 14:16 DOI 10.1186/s12916-016-0561-2 Galipeau et al. BMC Medicine (2016) 14:16 DOI 10.1186/s12916-016-0561-2 Background There are also individual websites and blogs, such as “Journalology” [8], that pro- vide thoughtful commentary on current issues related to publication science. Several commercial groups offer short training courses for editors [9, 10]. However, for any com- prehensive editor training program to work effectively and be assimilated into practice, it must be based on what the broader biomedical editor community considers to be core competencies. Unlike many other professional groups, such as clini- cians and healthcare professionals, many scientific edi- tors of biomedical journals operate largely without formal training and universal certification is not yet a high priority [5]. Instead, editors generally are invited to serve in their role by publishers, based on their expertise and stature in the field, since such expertise is essential for evaluating research and stature is important for es- tablishing the reputation of the journal and attracting submissions. However, such expertise does not guaran- tee that editors have the background or training neces- sary to carry out their roles and responsibilities. Editors may or may not be paid for their role and financial sup- port for the editorial role often does not include travel or training funds. Most editors work part-time as they continue their academic responsibilities in research and/or clinical work, with time for completing editorial responsibilities – much less training – being at a pre- mium. Researchers and peer reviewers similarly have no international standardized formal training or certifica- tion as to research conduct, reporting, and evaluation, making the editor’s job even more demanding. This situation is highly problematic given that the conse- quences of deciding what gets published and the degree of quality that is acceptable impacts future research, de- cisions, and healthcare directly. Our view is that the We are unaware of any body of literature systematic- ally identifying core competencies for biomedical editors, nor any agreement on or attempt at a consensus process to determine what they should be. For the purposes of this research, we borrow from the literature on competency-based continuing professional development to define competence as “the array of abilities across multiple domains or aspects of [practitioner] perform- ance in a certain context” [11]. We thus define core competencies as the essential knowledge, skills, and be- haviors necessary for the practice of scientific editing of biomedical journals. Background lack of consistent training of editors reduces the value of the published literature, including its quality, trans- parency, and reproducibility, thereby reducing value for money to funders and the usability of research findings, ultimately degrading public trust in the research record [3]. However, we are unaware of any research that dir- ectly addresses this topic. Additionally, while the train- ing of biomedical editors is an important mechanism to ensure the quality of the published literature, other im- portant changes in tandem with this, including re- examining the training offered to peer-reviewers and training graduate students in study conduct, analysis, interpretation, and reporting, could also have a benefi- cial effect. “…journals, some of which have reported research for many decades, are still not producing articles that are clear enough to really judge a study’s conduct, quality, and importance—let alone to allow other researchers to reproduce it or build on it” [1]. Biomedical journals are the main route for disseminat- ing the results of health-related research [2]. However, when examined more closely, the articles that journals publish are problematic; critical details are often missing or poorly reported, consequently reducing their quality, transparency, reproducibility, and usefulness for decision makers [3] – this is wasteful, diminishes scientific and fiscal value, and is unethical [4]. Authors and scientific journals share the majority of the responsibility for these shortcomings, as the former are accountable for the in- tegrity of a study’s conduct and the accuracy of reporting of the content within the manuscript, while the latter are accountable for decisions regarding its publication. On the side of journals, it is scientific editors (by which we mean editors, and ultimately the Editor-in-Chief, who are tasked with making decisions about the content and policies of journals) who are accountable for all ma- terial published in their journals. Readers have a right to expect these editors to implement all reasonable actions that could lead to best practices within their journals, as well as journals having processes in place to ensure the quality of the papers they publish. Some organizations, for example, the World Association of Medical Editors (WAME) [6] and the Committee on Publication Ethics (COPE) [7], provide rigorously- developed resources for biomedical journal editors free of charge, including guidance on the role of the medical edi- tor, editorial policies, and listservs on which editors’ ques- tions and issues are discussed. Background We believe it is important to de- velop a set of core competencies so that training pro- grams can then be developed and tailored with the intent that all editors meet some basic globally agreed- upon standards. Other stakeholders, such as publishers (including medical associations who have their own jour- nals), peer reviewers, and authors (researchers), also need to contribute to this effort. Herein, as a starting point, we report a scoping review of possible core com- petencies of scientific editors of biomedical journals. Abstract 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 17 Page 2 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Objectives The objective of this scoping review was to conduct a systematic search of the literature on the competencies Galipeau et al. BMC Medicine (2016) 14:16 Page 3 of 17 Page 3 of 17 journals, and an associated environmental scan that found only a few training opportunities for these editors [18]. Additionally, our anecdotal experience was that most descriptions of editor competencies are found in editorial-type publications (e.g. commentaries), job post- ings, and guidance documents (e.g. COPE Guidelines for Editors) – all of which would not generally be captured in a traditional scoping review of published research. required for scientific editors of biomedical journals to effectively and efficiently produce transparently reported and correctly analyzed and interpreted publications. Our specific aim was to answer the research question: “What is known from the literature on the competency require- ments of scientific editors of peer reviewed biomedical journals?” with the goal of summarizing the existing lit- erature. The purpose of this scoping review is to inform the future development of a set of core competencies for scientific editors of biomedical journals, which we hypothesize will ultimately lead to improvements in the quality of the published literature. The expanded scoping exercise included (1) searching the databases from the search strategy and grey litera- ture for non-research-based publications, (2) search- ing the results of an environmental scan from a previous related project [17, 18], (3) conducting a new environmental scan with additional search terms, and (4) searching the websites of existing networks (i.e. EQUATOR Network), major biomedical journal publishers (i.e. Wiley-Blackwell, Elsevier, BioMed Central, BMJ Publishing Group, Springer), and organizations that offer resources for editors (i.e. COPE, WAME, Council of Science Editors, European Association of Science Editors, and International Committee of Medical Journal Editors). For the database searches, the full-text of all potentially relevant documents were retrieved and independently reviewed for eligibility, in duplicate, by members of the team using a priori eligibility criteria. Disagreements between reviewers were resolved by consensus or by a third member of the research team. Both environmen- tal scans employed the same methodology, which involved the use of the Google search engine to run a series of two- and three-word keyword searches (Additional file 1). For each set of search outcomes, the first 50 Google results were screened for relevant information. If any of the last 10 results contained useful information, another 10 results were screened. Search strategy We searched the MEDLINE®, Cochrane Library, Embase®, CINAHL, PsycINFO, and ERIC databases, all from incep- tion to November 10th, 2014 (Additional file 1). The spe- cific search strategies were created by a Health Sciences Librarian with expertise in scoping review searching. The MEDLINE strategy was developed with input from the project team, then peer reviewed by a second librarian not otherwise associated with the project using the PRESS standard [15]. We also conducted a grey literature search, which included hand searching the reference lists of in- cluded articles, as well as searching key journals, in par- ticular JAMA from 1989, and BMJ, PLoS Medicine, European Science Editing, Annals of Internal Medicine, and CMAJ from 2004 onward to identify publications re- lated to the Peer Review Congresses [16]. Expanded scoping exercise Given that this scoping review is part of a larger pro- gram to develop core competencies for scientific editors of biomedical journals, in addition to seeking research literature, we also incorporated an expanded scoping exercise that included non-research-based (published and unpublished) materials to fill an expected gap in re- search on competencies of scientific editors of biomed- ical journals. This expectation was based on a previous systematic review [17], which found no comparative studies on training for scientific editors of biomedical Methods The protocol for this project has previously been depos- ited in the University of Ottawa’s Digital Repository (uOttawa Research) prior to beginning the screening phase [12]. Our methodological approach was guided by the Arksey and O’Malley Framework [13], as well as the additional suggestions to this framework made by Levac [14]. Specifically, we undertook the six-step process of: (1) identifying the research question; (2) identifying rele- vant studies; (3) study selection; (4) charting the data; (5) collating, summarizing, and reporting results; and (6) consultation. Objectives This process was continued until a set of 10 results with no relevant information was found [18]. Study selection: inclusion criteria Population Articles with statements mentioning competencies, knowledge, skills, behaviors, and tasks (henceforth re- ferred to as ‘competency-related statements’) pertaining to the role of scientific editors of peer-reviewed health- related journals (including Editors-in-Chief and associate/ academic editors, and full-time professional journal edi- tors) were included. Articles related to all other types of editor not directly involved in evaluation, peer review, re- vision, and selection of content (e.g. managing editors, technical editors, copy editors) were excluded. Disciplines We adopted MEDLINE’s journal selection criteria for our definition of health [19]. This definition includes journals that are “predominantly devoted to reporting original investigations in the biomedical and health Page 4 of 17 Page 4 of 17 Galipeau et al. BMC Medicine (2016) 14:16 sciences, including research in the basic sciences; clinical trials of therapeutic agents; effectiveness of diagnostic or therapeutic techniques; or studies relating to the be- havioral, epidemiological, or educational aspects of medicine” [19]. This definition encompasses biomedical journals as well as those in the disciplines of psychology and education. For feasibility purposes, we did not in- clude journals from the physical or natural sciences. sciences, including research in the basic sciences; clinical trials of therapeutic agents; effectiveness of diagnostic or therapeutic techniques; or studies relating to the be- havioral, epidemiological, or educational aspects of medicine” [19]. This definition encompasses biomedical journals as well as those in the disciplines of psychology and education. For feasibility purposes, we did not in- clude journals from the physical or natural sciences. scans we extracted the URL, title of the document, lan- guage of publication, and who produced the document (affiliation). For all documents, we collected descriptions of any statements potentially relating to the competen- cies of scientific editors of biomedical journals, such as descriptions of particular skills, knowledge, attitudes, behaviors, tasks, and training. Collating, summarizing, and reporting the results In an effort to create a useful summary of the data for the next steps of our program to develop core compe- tencies for scientific editors of biomedical journals, we combined the competency-related statements retrieved from all sources. First, two people (JG, KDC) classified all statements pertaining only to Editors-in-Chief into a single category, since these would be considered to be beyond the core competencies of scientific editors more generally. They then collated overlapping or duplicate statements to produce a list of unique statements. Fi- nally, they grouped statements into emergent themes to make them more manageable for future use (e.g. in an upcoming Delphi exercise), and so that they would be understandable to readers. While some of the wording of particular statements was modified to assimilate over- lapping statements, where statements were expressed as knowledge, skills, behaviors, or tasks that implied com- petencies, but not as competencies themselves, we did not edit or translate them to express competencies, in order to preserve the original intent. Charting the data A data extraction form was developed a priori to capture information on each document included in the review. It was piloted and refined based on feedback from the ex- ercise. Three people (JG, KDC, LS) carried out data ex- traction in the following manner: data were extracted by one reviewer and a second reviewer conducted verifica- tion of the data for all records. Disagreements between reviewers were resolved by consensus. General study characteristics extracted for the database search were: first author name and contact information (of corre- sponding author), year of publication, institutional affili- ation of first author, country, language of publication, study design, and funding source. For the environmental Database search We screened 5,837 titles and abstracts, of which 360 were screened in full-text (Fig. 1). Of these, 206 were ex- cluded, leaving 154 publications meeting the inclusion criteria. Twenty-five of these publications were research based (Table 1) and the remaining 136 were editorial in nature (Additional file 2). Disciplines The relationships between behaviors, tasks, and competencies will be the subject of discussion and translation undertaken as part of the consensus meeting phase of this project. Screening Following the execution of the search strategy, the iden- tified records (titles and available abstracts) were collated in a Reference Manager [20] database for de- duplication. The final unique record set of potentially eligible studies was exported to Internet-based soft- ware, DistillerSR (Evidence Partners, Ottawa, Canada), through which screening of records and data extrac- tion were carried out. Each title and abstract was screened by two of four reviewers (LS, JG, JT (see Acknowledgements), and MG (see Acknowledgements)) using a ‘liberal accelerated’ method [21] (i.e. one reviewer screened each record and a second reviewer screened only excluded records, independently). The full-text of all remaining potentially eligible papers was then retrieved, uploaded into DistillerSR, and reviewed for eligibility, in- dependently, by two members of the team (LS and JG) using a priori eligibility criteria. Disagreements between reviewers at this stage were resolved by consensus or by a third member of the research team. Study designs Th The review included all study designs as well as edito- rials and commentaries. Economic evaluations and letters were excluded, as neither was expected to con- tribute useful data for the purposes of this scoping review. For feasibility purposes, we included articles written in English and French only. We did not include English or French abstracts of papers written in another language. Charting the data General characteristics Research-based publications A total of 18 publications from the database search presenting the results of re- search (subsequently termed ‘research-based publica- tions’) were considered relevant to core competencies for scientific editors of biomedical journals, along with another seven articles found in the grey literature search (including six conference abstracts for which there ap- pears to be no full text publication) (Table 1). None of these 25 articles had an outline or description of core competencies of scientific editors as an objective of the Galipeau et al. BMC Medicine (2016) 14:16 Page 5 of 17 Fig. 1 Study flow diagram scientific editors. Overall, 133 publications were editorial in nature, while the remaining three included a lecture, a job description, and an interview. Seventeen journals had multiple included publications, with three of them (Australian and New Zealand Journal of Obstetrics and Gynaecology, Croatian Medical Journal, and Indian Journal of Medical Sciences) producing three publica- tions each while the other 14 journals had two publica- tions each. JAMA also had four publications across three journals in its collection (JAMA, JAMA Internal Medicine, JAMA Ophthalmology). The rest of the sample consisted of a single publication per journal. The date of publication ranged from 1954 to 2015; 2011 (n = 20) and 2012 (n = 17) were the 2 years with the most studies. The sample included 66 studies with first au- thors originating from the USA and 19 from the United Kingdom, with representation from another 18 countries as well among first authors. The individual publications yielded between zero and 15 competency- related statements, with a median of two statements per publication. research. Fifteen studies were survey-based research, three were descriptive studies, three were case studies, and two were final project reports (one from a task force and the other from a study funded by the US National Institute of Education). The remaining two studies were a systematic review and a mixed-methods study. Five studies reported receiving funding. Nineteen studies were published in different journals and the remaining six were part of the Peer Review Congress Proceedings. Publication dates ranged from 1975 to 2014; 2009 was the year with the most publications (n = 3), followed by 2011 (n = 2) and 2013 (n = 2). Twelve of the studies’ first authors were from the USA, 10 from the United Kingdom, and one each from Iran, India, and Canada. Charting the data General characteristics The pub- lications produced a total of 40 competency-related statements (i.e. possible competencies), with individual publications yielding between zero and 14 statements and a median of one statement per publication. Expanded scoping exercise Non research-based publications A total of 136 non- research-based publications were considered relevant to core competencies for scientific editors of biomedical journals, yielding a total of 537 competency-related statements (Additional file 2). Similar to the research- based literature, none of these publications had the ex- plicit goal of outlining a set of core competencies for Environmental scan of training in Journalology We reviewed all 258 documents listed in the Repository of Ongoing Training Opportunities in Journalology [20], which houses all of the data from an environmental scan of training in Journalology carried out by members of Galipeau et al. BMC Medicine (2016) 14:16 Page 6 of 17 Table 1 Included research-based publications First author Affiliation Country Journal Year Design # a Item(s) b Albert, T Tim Albert Training UK Learned Publishing 2002 Survey 0 N/A Barnes, M University of Nebraska-Lincoln USA The Review of Higher Education 1986 Survey 5 5, 72, 170, 171, 185 Carroll-Johnson, R Oncology Nursing Society USA Nurse Author & Editor 1996 Survey 1 137 Davis, RM Henry Ford Health System USA Science & Engineering Ethics 2002 Survey 1 137 de Jesus Mari, J King’s College, University of London UK African Journal of Psychiatry 2009 Task Force Report 0 N/A Etemadi, A Shaheed Beheshti University of Medical Sciences Iran Saudi Medical Journal 2004 Survey 1 141 Freda, M Journal of Nursing Scholarship USA Journal of Nursing Scholarship 2005 Survey 0 N/A Froehle, T Indiana University, Bloomington USA Counselor Education & Supervision 1990 Descriptive Study 0 N/A Galipeau, J Ottawa Hospital Research Institute Canada Systematic Reviews 2013 Systematic Review 0 N/A Garrow, J LOCKNET Peer Review Research Group: European Journal of Clinical Nutrition UK Journal of the American Medical Association 1998 Survey 0 N/A Grindlay, D Centre for Evidence-based Veterinary Medicine, School of Veterinary Medicine and Science, The University of Nottingham UK BMC Veterinary Research 2014 Survey 1 148 Hing, C Department of Trauma & Orthopaedics, St George’s Hospital, Tooting, UK UK Journal of Orthopaedic Surgery & Research 2011 Survey 0 N/A Kearney, M University of Rochester School of Nursing USA Nursing Outlook 2006 Descriptive Study 13 5, 26, 30, 35, 69, 72, 83, 84. Expanded scoping exercise 85, 91, 102, 178, 203 Kleinert, S The Lancet UK Peer Review Congress (Abstract) 2005 Observational Study 0 N/A Lebeau, DL Tulane University Medical Center USA Peer Review Congress (Abstract) 1997 Survey 1 72 Logothetti, H Obstetrics & Gynecology USA Peer Review Congress (Abstract) 2009 Case-Control 1 101 Patrone, D Philosophy Department, State University of New York at Oneonta/Broome Community College, Binghamton, New York USA Biosecurity and Bioterrorism: Biodefense Strategy, Practice, and Science 2012 Survey 2 147, 203 Radford, D Division of Prosthetic Dentistry, King’s and St Thomas’ Dental Institute, London UK British Dental Journal 1999 Survey 0 N/A Reynolds, T Highland Hospital USA Peer Review Congress (Abstract) 2009 Survey 0 N/A Silverman, R Ohio State University USA None (Final Report) 1975 Final Report 6 79, 91, 101, 166, 194, 203 Srinivasan, S Indian J Medical Ethics India Peer Review Congress (Abstract) 2013 Survey 0 N/A Wager, E Sideview, Princes Risborough UK Peer Review Congress (Abstract) 2009 Case Analysis 0 N/A Wager, E Sideview, Princes Risborough UK The British Medical Journal 2013 Quantitative + Interviews 1 136 Williams, P University College London UK Science and Engineering Ethics 2011 Case Studies 2 58, 203 Wong, V Department of Neurology, University of Michigan, Ann Arbor USA Journal of Clinical Epidemiology 2011 Survey 5 2, 118, 123, 203(2) TOTAL 40 a Number of competency statements extracted from the document b Corresponds to the item number from the list of competency-related statements (Table 3) a Number of competency statements extracted from the document b Corresponds to the item number from the list of competency-related statements (Table 3) a Number of competency statements extracted from the document b Corresponds to the item number from the list of competency-related statements (Table 3) relating to scientific editors of biomedical journals (Table 2). Seven of these documents were from organiza- tions that provide guidance to editors (e.g. WAME, our team in 2013. From this repository, we extracted 11 relevant non-research-based articles from which we were able to retrieve 556 competency-related statements Page 7 of 17 Galipeau et al. Expanded scoping exercise BMC Medicine (2016) 14:16 Table 2 Included documents from expanded scoping exercise GUIDANCE FROM EDITORIAL GROUPS Title Editorial group/organization Scanning source # a Item(s) b International Standards for Authors Committee on Publication Ethics (COPE) Training 58 6(2), 9, 10, 11, 12(2), 20(6), 22(3), 25, 26, 34, 35(3), 39, 46, 57(2), 64, 72(5), 79(2), 108(2), 137(2), 138(4), 140, 141(4), 143, 144, 145, 146(3), 150, 159, 198, 199, 203 Guidelines for Editors COPE Training 69 2, 6, 16, 19, 20, 21,22(2), 24, 25, 26(2), 27, 28, 30(2), 32, 34, 35(4), 39, 41, 42, 46, 54, 61, 67, 72(6), 79(2), 80, 83, 87(2), 88, 104, 111, 137(2), 138, 139, 141(2), 143, 146, 150, 153(2), 159, 189, 194, 197, 198, 199(2), 203(7) A Short Guide to Ethical Editing for New Editors COPE Training 34 6(2), 10, 20(3), 22, 26(2), 30, 32, 35(2), 36, 48(2), 62, 68, 72, 104, 116, 136, 137(2), 138, 144(2), 189, 197, 203(5) A Science Editing Course for Graduate Students Council of Science Editors (CSE) Core competencies 6 5, 95, 98, 135, 138, 178 Can Non-Native-English-Speaking Editors be Effective Editors of English-Language Writing? Alliance of Crop, Soil, and Environmental Sciences Society Expanded scoping exercise CSE Core competencies 13 25, 78, 95, 100(2), 126(2), 127, 132, 138, 141, 178, 199 Guidelines for Reporting Health Research: How to Promote their Use in Your Journal EQUATOR Network Networks search 0 N/A Research Ethics, Publication Ethics and Good Practice Guidelines EQUATOR Network Networks search 0 N/A European Association of Science Editors (EASE) Toolkit for Journal Editors EASE Core competencies 0 N/A Editor’s Handbook (2nd Edition) EASE Core competencies 166 2(2), 7, 8, 9, 10, 15(2), 16, 18, 20(2), 22(4), 24(2), 25, 26(2), 27(2), 28, 30, 31, 32 33 35(3) 36 39(2) 48 50(3) 52(2) 201(3), 202(2), 203(8) Core competencies 10 6, 15, 35, 72(2), 85, 89, 111, 138, 149 Core competencies 2 35, 140 Training 9 1, 13, 38, 46, 47(2), 140(3) Training 56 6, 10, 14, 18, 20(4), 26(3), 29, 35, 43, 47, 48(2), 52, 57(6), 61(2), 62, 72(2), 79, 83, 84, 91, 92, 106, 113, 119, 137, 139, 141(2), 146(2), 159, 189, 197(2), 203(9) Training 84 17, 20(2), 22, 26(5), 33, 37, 48, 50, 51, 52, 56(2), 59, 61, 64(3), 65(3), 66, 68(5), 70, 72, 74, 75, 76, 80, 81, 84, 90, 91(2), 106, 109, 113(4), 124(3), 127, 128, 132, 133(2), 135(2), 136, 137, 140, 141(5), 142, 144, 145, 146, 152, 154, 155(3), 159, 162(2), 166, 198, 203(4) Core competencies 0 N/A Core competencies 10 6, 15, 35, 72(2), 85, 89, 111, 138, 149 Core competencies 2 35, 140 ASSOCIATIONS, JOURNALS, PUBLISHERS Editor Handbook Alliance of Crop, Soil, and Environmental Sciences Society ASSOCIATIONS, JOURNALS, PUBLISHERS Editor Handbook Alliance of Crop, Soil, and Environmental Sciences Society Core competencies 0 N/A Alliance of Crop, Soil, and Environmental Sciences Society Galipeau et al. Expanded scoping exercise BMC Medicine (2016) 14:16 Page 8 of 17 Table 2 Included documents from expanded scoping exercise (Continued) Table 2 Included documents from expanded scoping exercise (Continued) Editors and Reviewers Alliance of Crop, Soil, and Environmental Sciences Society Core competencies 11 6(3), 35(2), 47, 61, 112(2), 140, 141 Editor Handbook American Association of Pharmaceutical Scientists Journal Core competencies 9 35, 91, 116(2), 203(5) Editor-In-Chief: Position Description American Geophysical Union Core competencies 25 69, 72, 84, 95, 140, 159, 160, 161, 162(2), 166, 167, 170(2), 171(2), 178, 181(2), 182, 189, 191(2), 199, 203 Position Description for the AJNR Editor and Basic Qualifications American Journal of Neuroradiology Core competencies 5 5, 70, 91, 117, 168 Responsibilities of an Editor Annals, Academy of Medicine, Singapore Training 30 6(2), 17, 19, 35(2), 61, 73, 77, 79, 80, 81, 83, 90(4), 91, 92, 137(4), 138, 151, 172, 192, 203(3) What does an Associate Editor Actually do? Association for Computing Machinery Core competencies 3 34, 40, 72 The Role of the Scientific Editor British Dental Journal Core competencies 3 8, 14, 20 Recruiting a Journal Editor: An HSS Challenge Cambridge Journals Blog Core competencies 0 N/A Editorial: on Editing and Being an Editor Cultural Studies of Science Education Core competencies 3 6, 33, 34 Editor’s Pack Elsevier Publishers search 0 N/A How do Publishers Choose Editors, and How do they Work Together? Expanded scoping exercise ResearchGate Core competencies 5 44, 72, 79, 159, 203 Job Description of an Editor-in-Chief Study.com Core competencies 0 N/A So, you want to be a Science Writer when you grow up… The Black Hole Core competencies 0 N/A Ideas for a Topical Outline Unknown Training 103 2, 8(2), 10, 14, 15, 17, 20, 22, 26(3), 31, 33, 37, 48, 49, 50, 51, 52, 53, 56(2), 57, 59(2), 61(2), 62, 64(3), 66, 68(4), 70, 71, 72, 74, 75, 76, 77(2), 81, 82, 91(3), 92, 95, 101, 106(2), 113(3), 117, 124(3), 127, 133(4), 135(2), 136, 137, 140, 141(3), 142, 144, 145, 146, 147, 152, 154, 155(3), 161, 162(2), 165(2), 169(2), 173, 198, 203(9) Recommended Recruitment Steps for Journal Editor, CJNSE/RCJCÉ Wired Learning Consultants Core competencies 6 95, 117, 162, 167, 178(2) Table 2 Included documents from expanded scoping exercise (Continued) Trainee Programs/Editorial Trainees Springer Core competencies 0 N/A Confessions of a Journal Editor The Chronicles of Higher Education Core competencies 0 N/A Ethics and the Psychiatry Journal Editor: Responsibilities and Dilemmas The Israel Journal of Psychiatry and Related Sciences Core competencies 9 20(2), 22, 36, 48, 64, 120, 140, 141 Editor Ethics 2.0 Code/Affirming Editors University of North Carolina – Charlotte Core competencies 2 20, 138 UOJM Editor Training: Results from the 2013 Editor Satisfaction Survey and Highlights from 2013–2014 Training Workshops University of Ottawa Journal of Medicine Core competencies 8 36, 61, 74, 79, 95, 123, 160, 175 FAME Guidelines World Health Organization Training 35 6(3), 10, 14, 15, 30, 34, 35, 39, 40(2), 47, 61, 72(3), 75, 79(2), 91, 92, 133, Table 2 Included documents from expanded scoping exercise (Continued) Research Journal Editor Position Description Young Adult Library Service Associa OTHER SOURCES Medical/Scientific Editor (job posting) Alexion Pharmaceuticals, Inc. Duties of Editors Bioinfo Publications What is Special about Science Editing? Biotext What is Different about Science Editing? Emend Editing What Exactly Does an Editor Do? Expanded scoping exercise Elsevier Core competencies 7 110, 160, 162, 184, 185, 194, 203 Publishing Ethics Resource Kit (PERK) Elsevier Publisher search 0 N/A European Respiratory Journal Editor(s)-in-Chief European Respiratory Society Core competencies 6 162(2), 165, 167, 195, 178 Editor-in-Chief (position description) International Society of Exposure Science; Journal of Exposure Science and Environmental Epidemiology Core competencies 9 19, 35, 101, 138, 159, 165, 166, 170, 203 Editor-in-Chief (position description) Journal of Family Planning and Reproductive Health Care Core competencies 19 8, 19(2), 36, 95, 116, 117, 121, 123(2), 127, 138, 159, 164, 167, 170, 178, 180, 181 Responsibilities of the Editor Journal of Medical Internet Research Core competencies 1 123 Responsibilities of the JNCI Editor‐in‐Chief Journal of the National Cancer Institute Core competencies 8 95, 99, 127, 132, 138, 159, 174, 203 Responsibilities of Editors and Reviewers Online Ethics Center for Engineering and Science Core competencies 0 N/A Scientific Editing–A Wise Career Choice Science Careers (from the journal Science) Core competencies 0 N/A Horses for Courses–Research Papers versus Reviews Science Careers (from the journal Science) Core competencies 0 N/A The Editors' World: Back to the Books Science Careers (from the journal Science) Core competencies 6 159(2), 165, 175, 178, 181 Bench to Page: An Editor's View of Science Publishing Science Careers (from the journal Science) Core competencies 3 73, 164, 170 At the Gateway of Cutting-Edge Research Science Careers (from the journal Science) Core competencies 7 68, 108, 109, 122, 162, 163, 191 Translating Scientific Expertise into Publishing Success Science Careers (from the journal Science) Core competencies 0 N/A Journal Editors Get Twitter-Savvy Science Careers (from the journal Science) Core competencies 14 40, 71, 73, 90, 93, 95, 108, 164, 165, 174, 177, 185, 193, 196 Careers in Neuroscience/Career Paths: Science Publishing Society for Neuroscience Core competencies 8 122, 164, 165, 166, 167(2), 178, 184 Academic Journal Editors’ Professionalism: Perceptions of Power, Proficiency and Personal Agendas Society for Research Into Higher Education Core competencies 9 33(2), 72, 73, 110, 121, 160, 167, 174 Editorial Guide Springer Publishers search 7 26, 65, 78, 82, 85, 138, 197 Springer Galipeau et al. Expanded scoping exercise BMC Medicine (2016) 14:16 Page 9 of 17 Table 2 Included documents from expanded scoping exercise (Continued) Trainee Programs/Editorial Trainees Springer Core competencies 0 N/A Confessions of a Journal Editor The Chronicles of Higher Education Core competencies 0 N/A Ethics and the Psychiatry Journal Editor: Responsibilities and Dilemmas The Israel Journal of Psychiatry and Related Sciences Core competencies 9 20(2), 22, 36, 48, 64, 120, 140, 141 Editor Ethics 2.0 Code/Affirming Editors University of North Carolina – Charlotte Core competencies 2 20, 138 UOJM Editor Training: Results from the 2013 Editor Satisfaction Survey and Highlights from 2013–2014 Training Workshops University of Ottawa Journal of Medicine Core competencies 8 36, 61, 74, 79, 95, 123, 160, 175 FAME Guidelines World Health Organization Training 35 6(3), 10, 14, 15, 30, 34, 35, 39, 40(2), 47, 61, 72(3), 75, 79(2), 91, 92, 133, 138(2), 141, 143, 149, 159(2), 179, 189, 198, 203(2) Research Journal Editor Position Description Young Adult Library Service Association Core competencies 11 35, 95(2), 117, 159, 160, 161, 162, 167, 174, 180, OTHER SOURCES Medical/Scientific Editor (job posting) Alexion Pharmaceuticals, Inc. Core competencies 0 N/A Duties of Editors Bioinfo Publications Core competencies 3 104, 141, 199 What is Special about Science Editing? Biotext Core competencies 11 120, 124, 125, 129(2), 130, 131, 141, 165, 175, 193 What is Different about Science Editing? Emend Editing Core competencies 13 57, 60, 117, 125, 126, 127, 129, 130, 131, 162, 165, 175, 193 What Exactly Does an Editor Do? Joseph Alpert Training 11 39, 54, 68, 73, 86, 108, 138, 162, 198, 203(2) Becoming a Journal Editor PhD2Published Core competencies 5 109, 167, 182, 190, 199 Public Knowledge Project School Public Knowledge Project Training 67 6(3), 14, 15(8), 20, 26(3), 30, 34, 35(3), 39, 46, 47, 50, 53(3), 57(2), 59, 61(2), 62, 68, 84, 85, 87, 91, 95, 104(2), 137(2), 138, 140, 141, 149, 160, 167(2), 197(2), 203(14) What does an Editor (a Member of Editorial Board) do Exactly in Journals? ResearchGate Core competencies 0 N/A What are the Role and Duties of a Scientific Editor of an Academic Peer-Review Journal? Expanded scoping exercise Joseph Alpert Becoming a Journal Editor PhD2Published Public Knowledge Project School Public Knowledge Project Core competencies 0 N/A Core competencies 3 104, 141, 199 Core competencies 11 120, 124, 125, 129(2), 130, 131, 141, 165, 175, 193 Core competencies 13 57, 60, 117, 125, 126, 127, 129, 130, 131, 162, 165, 175, 193 Training 11 39, 54, 68, 73, 86, 108, 138, 162, 198, 203(2) Core competencies 5 109, 167, 182, 190, 199 Training 67 6(3), 14, 15(8), 20, 26(3), 30, 34, 35(3), 39, 46, 47, 50, 53(3), 57(2), 59, 61(2), 62, 68, 84, 85, 87, 91, 95, 104(2), 137(2), 138, 140, 141, 149, 160, 167(2), 197(2), 203(14) Core competencies 0 N/A Core competencies 5 44, 72, 79, 159, 203 Core competencies 0 N/A Core competencies 0 N/A Training 103 2, 8(2), 10, 14, 15, 17, 20, 22, 26(3), 31, 33, 37, 48, 49, 50, 51, 52, 53, 56(2), 57, 59(2), 61(2), 62, 64(3), 66, 68(4), 70, 71, 72, 74, 75, 76, 77(2), 81, 82, 91(3), 92, 95, 101, 106(2), 113(3), 117, 124(3), 127, 133(4), 135(2), 136, 137, 140, 141(3), 142, 144, 145, 146, 147, 152, 154, 155(3), 161, 162(2), 165(2), 169(2), 173, 198, 203(9) Core competencies 6 95, 117, 162, 167, 178(2) Core competencies 0 N/A What does an Editor (a Member of Editorial Board) do Exactly in Journals? ResearchGate What are the Role and Duties of a Scientific Editor of an Academic Peer-Review Journal? ResearchGate Job Description of an Editor-in-Chief Study.com So, you want to be a Science Writer when you grow up… The Black Hole Ideas for a Topical Outline Unknown Core competencies 0 N/A Number of competency statements extracted from the document p y b Corresponds to the item number from the list of competency-related statements (Table 3) Galipeau et al. BMC Medicine (2016) 14:16 Page 10 of 17 Page 10 of 17 Page 10 of 17 COPE, International Committee of Medical Journal Editors) and the remaining documents were from a variety of other sources. group of editors will collaborate to outline a set of core competencies. Despite our finding that the competencies of editors have been discussed in the published literature since the mid-1950s, a recent systematic review [17] found no comparative studies of the effectiveness of training for editors. This is concerning, given the gate-keeping role that scientific editors play as guardians of the scientific record [23]. Expanded scoping exercise Trends in the number of publications annu- ally would seem to indicate that, while the overall num- ber of publications in this area has grown since the topic first emerged in the literature, interest peaked around 2011 and is now waning. The trend, however, is more reflective of editorial-type articles in journals, which have declined since 2011, while the number of research- based publications has remained relatively stable (yet sparse) during the same timeframe. Environmental scan of core competencies for scien- tific editors of biomedical journals The environmental scan carried out for this project consisted of a total of 40 keyword searches (Additional file 1) that yielded 48 rele- vant documents, of which 35 were deemed to meet the eligibility criteria after screening in duplicate. These 35 documents produced 426 competency-related state- ments (Table 2). Among the sample, 18 documents were produced or published by journals, nine were from associations and societies, six were from organizations providing guidance to editors, and two were from publishers. Search of networks, publishers, and meetings A search of networks, publishers, and meetings produced an additional five documents, three of which were docu- ments from publishers’ websites and two from the EQUATOR Network (Table 2). We extracted seven competency-related statements from these additional documents. One possibility for the decline in total publications is that organizations may believe these issues have already been identified and they are working on training mate- rials for them rather than publishing more research or commentaries about them in medical journals. A possi- bility for the continued lack of research in this area may be that the focus of many major funding agencies is to fund by disease (e.g. heart, kidney, cancer, diabetes). As journal editing, and the field of journalology more broadly, is a domain that spans across the spectrum of research on diseases, it is often difficult to find appropri- ate funding opportunities and even more difficult to convince specific disease-based funding agencies that the research merits their funding investment. Of the 64 documents in the expanded scoping exer- cise, the European Association of Science Editors Edi- tor’s Handbook (2nd Edition) [22] had the highest number of competency-related statements (n = 166). The median number of competency-related statements across the scoping exercise documents was six. Collating and summarizing the data A major challenge that we anticipated as part of this scoping review was that a large proportion of the evi- dence may not be in the traditional research-based lit- erature. For this reason, we placed a heavy emphasis on extensively searching the grey and non-research-based literature by including two related environmental scans as part of the larger program on core competencies for scientific editors of biomedical journals. However, it is still possible that we may not have captured every aspect of a biomedical editor’s work in our searches. In particu- lar, some of the more tacit (e.g. difficult to describe) as- pects of the work of editors may simply not be documented, or may reside in documents that may not necessarily be found in a database or Google search (e.g. resources residing behind membership or password- protected webpages, paywalls). We expect that any crit- ical missing items will be brought forth in the training needs assessment, the Delphi, or the consensus meeting. Additionally, because of the subjective nature of this type of data extraction, it is possible that some competency-related statements were missed within the included publications. However, we feel that the likeli- hood of this was reduced as several members of the The combined 1,566 competency-related statements ori- ginating from the 225 total documents were collated and then de-duplicated, producing a list of 203 unique state- ments (i.e. possible competencies of scientific editors) (Table 3). The statements were organized into seven categories that emerged from the data: (1) dealing with authors; (2) dealing with peer reviewers; (3) journal pub- lishing; (4) journal promotion; (5) editing; (6) ethics and integrity; and (7) qualities and characteristics of editors. Discussion This scoping review identified 225 relevant publications, spanning more than 60 years, and involving authors from more than 20 countries. It produced a comprehen- sive list of possible competency-related statements for the scientific editor position within biomedical journals. This categorized list of statements will be used in a sub- sequent Delphi exercise aiming to ask a broad spectrum of scientific editors of biomedical journals to rate the importance of each statement in relation to performing their duties as a scientific editor. This data will then help inform a consensus meeting in which a select Page 11 of 17 Galipeau et al. Discussion BMC Medicine (2016) 14:16 Scientific editors should: 1 Review study protocols and methods and encourage authors to make them publicly available 2 Ensure authors are aware of ethical authorship practices 3 Seek to help authors understand magnitude of effect 4 Assist potential authors in developing a spirit of inquiry 5 Develop wide acquaintance with potential authors 6 Demonstrate accountability to authors and ensure they are treated with fairness, courtesy, and objectivity 7 Provide constructive criticism to authors 8 Engage in mentorship and education of authors to help them produce work to best effect 9 Mediate sound communication between the comments of reviewers and responses of authors 10 Ensure publication decisions are clearly communicated to all authors 11 Interact with authors to confirm undisputed changes in authorship and act on any institutional findings concerning authorship disputes 12 Clarify the peer-review processes to authors 13 Negotiate manuscript publication delays with authors 14 Deal with authors who appeal against rejection 15 Ensure authors are informed about journal and article information and/or funding 16 Ensure that requests from authors that an individual not review their submission are respected, if these are well-reasoned 17 Engage in critical evaluation of authors’ manuscripts and the peer-review process itself 18 Provide active encouragement for revisions of manuscripts 19 Demonstrate experience as a competent author, academic, researcher, or reviewer 20 Demonstrate proficiency in dealing with author misconduct and other issues related to publication ethics 21 Work with publishers to defend author rights and pursue offenders 22 Act on concerns about plagiarism, data fabrication, or an authorship issue and follow-up with authors and then institutions 23 Request full disclosure of potential conflicts of interest by the authors 24 Support authors in dealing with breaches of copyright and plagiarism issues 25 Request appropriate documentation from authors when they submit manuscripts II. Discussion Dealing with peer reviewers 26 Develop, facilitate, and monitor the peer review process 27 Knowledge of different types of peer review 28 Encourage and demonstrate awareness of new findings on peer review and publishing and how these influence their journal’s processes 29 Review revised manuscripts 30 Provide guidance to peer reviewers 31 Ensure thorough statistical review 32 Ensure that peer review panels for individual papers are not biased 33 Evaluate and provide feedback to the reviewers on review quality 34 Ensure manuscript content is matched with the expertise of particular reviewers 35 Monitor and ensure the fairness, timeliness, thoroughness, and civility in the processing of manuscripts and in responding to queries from authors and reviewers 36 Demonstrate knowledge of the workings of the peer review process 37 Train peer reviewers 38 Ensure reviewer comments are shared with all peer reviewers 39 Synthesize reviews and make ultimate editorial decisions in light of peer reviewers’ comments 40 Evaluate manuscripts in light of reviewers’ critiques and various selection criteria Page 12 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Table 3 Competency-related statements a (Continued) 41 Demonstrate the ability to distinguish between objective peer reviewed research and reviews from opinion and the journal c from advertising and other promotional content 42 Ensure reviewers who consistently produce discourteous, poor quality, or late reviews are removed from the journal’s pool of reviewers 43 Ensure a decision is made on a manuscript when reviewers fail to submit a timely review 44 Ensure a very high standard of the referees, don’t accept sloppy reports from anyone 45 Demonstrate publication and reviewing skills and experience 46 Ensure that reviewers keep manuscripts, associated material, and the information they contain strictly confidential 47 Demonstrate sound judgment in the acceptance of research articles, editorials, and reviews that touch on current issues III. Discussion Journal publishing 48 Demonstrate knowledge of marketing and advertising policies, including ethical issues 49 Demonstrate knowledge of the article embargo process 50 Demonstrate knowledge of indexing services 51 Demonstrate knowledge of reprint processes 52 Demonstrate knowledge of the specifications of the journal 53 Demonstrate knowledge of the goals of the journal 54 Demonstrate knowledge of formatting of layout for journal issues 55 Ensure the content of manuscripts submitted for publication is checked for accuracy 56 Demonstrate knowledge of the different parts, purposes, and characteristics of different types of journals 57 Demonstrate understanding of the editorial office and operations 58 Ensure that selected/published research is correct 59 Demonstrate knowledge about legal issues relating to the position of scientific editor 60 Be aware of how design can be used to improve the readability of a document 61 Demonstrate understanding of one’s responsibilities and rights as a journal editor 62 Demonstrate knowledge of the roles and responsibilities of the editorial staff 63 Identify and address issues related to data protection and confidentiality 64 Demonstrate knowledge of journal metrics and research impact 65 Demonstrate knowledge of online publishing and products 66 Demonstrate knowledge of the parts, purposes, and characteristics of audio and video clips 67 Demonstrate awareness of intellectual property issues and work with publisher to handle potential breaches 68 Demonstrate knowledge of technical-economical aspects of medical journal production 69 Explore and embrace innovative technologies 70 Maintain close contact with the latest trends in electronic media (e.g. tablets) 71 Engage in multimedia publishing practices 72 Act as a gatekeeper and guarantor of publications, checking both the quality and scope of research published in the journal IV. Discussion Journal promotion 73 Maintain knowledge of important developments and trends in one’s own field 74 Demonstrate knowledge of history of journals and scientific publications 75 Demonstrate knowledge of national and regional variations between journals 76 Demonstrate knowledge of political and geopolitical issues 77 Demonstrate familiarity with associations and their educational resources 78 Stay on top of updates in one’s field 79 Demonstrate knowledge of, and work to maintain and improve the journal’s policies, vision, scope, content, processes, and g 80 Ensure decisions are based on the validity of the work and its importance to the journal’s readers Table 3 Competency-related statements a (Continued) 41 Demonstrate the ability to distinguish between objective peer reviewed research and from advertising and other promotional content 42 Ensure reviewers who consistently produce discourteous, poor quality, or late reviews reviewers 43 Ensure a decision is made on a manuscript when reviewers fail to submit a timely revi 44 Ensure a very high standard of the referees, don’t accept sloppy reports from anyone 45 Demonstrate publication and reviewing skills and experience 46 Ensure that reviewers keep manuscripts, associated material, and the information they 47 Demonstrate sound judgment in the acceptance of research articles, editorials, and rev III. Discussion Journal publishing 48 Demonstrate knowledge of marketing and advertising policies, including ethical issues 49 Demonstrate knowledge of the article embargo process 50 Demonstrate knowledge of indexing services 51 Demonstrate knowledge of reprint processes 52 Demonstrate knowledge of the specifications of the journal 53 Demonstrate knowledge of the goals of the journal 54 Demonstrate knowledge of formatting of layout for journal issues 55 Ensure the content of manuscripts submitted for publication is checked for accuracy 56 Demonstrate knowledge of the different parts, purposes, and characteristics of differen 57 Demonstrate understanding of the editorial office and operations 58 Ensure that selected/published research is correct 59 Demonstrate knowledge about legal issues relating to the position of scientific editor 60 Be aware of how design can be used to improve the readability of a document 61 Demonstrate understanding of one’s responsibilities and rights as a journal editor 62 Demonstrate knowledge of the roles and responsibilities of the editorial staff 63 Identify and address issues related to data protection and confidentiality 64 Demonstrate knowledge of journal metrics and research impact 65 Demonstrate knowledge of online publishing and products 66 Demonstrate knowledge of the parts, purposes, and characteristics of audio and video 67 Demonstrate awareness of intellectual property issues and work with publisher to han 68 Demonstrate knowledge of technical-economical aspects of medical journal productio 69 Explore and embrace innovative technologies 70 Maintain close contact with the latest trends in electronic media (e.g. tablets) 71 Engage in multimedia publishing practices 72 Act as a gatekeeper and guarantor of publications, checking both the quality and scop IV. Journal promotion 73 Maintain knowledge of important developments and trends in one’s own field 74 Demonstrate knowledge of history of journals and scientific publications 75 Demonstrate knowledge of national and regional variations between journals 76 Demonstrate knowledge of political and geopolitical issues 77 Demonstrate familiarity with associations and their educational resources 78 Stay on top of updates in one’s field 79 Demonstrate knowledge of, and work to maintain and improve the journal’s policies, v 80 Ensure decisions are based on the validity of the work and its importance to the journ 81 Ensure controversial topics (political, ethical) are dealt with 82 Stimulate others to write articles and editorials Page 13 of 17 Galipeau et al. Discussion BMC Medicine (2016) 14:16 Table 3 Competency-related statements a (Continued) Table 3 Competency-related statements a (Continued) 83 Engage in the promotion of scholarly research and best practices in conducting and reporting it 84 Entice leading researchers to submit to the journal 85 Serve as ambassador for the journal in establishing its visibility and image 86 Motivate physicians to read, ponder, and implement the information provided 87 Seek feedback/opinions on the journal 88 Enhance public understanding of science 89 Demonstrate understanding of who one’s constituency is 90 Demonstrate a responsibility to the scientific community 91 Hold paramount the interests of the particular journal’s readers 92 Engage in communication with the public 93 Engage with existing and new scientific communities V. Editing 94 Demonstrate knowledge of policies for submission of manuscripts 95 Demonstrate broad and detailed knowledge of the skills needed to refine a piece of scientific work and shepherd it through to publication 96 Demonstrate knowledge of typography 97 Demonstrate knowledge of and experience with online editing 98 Demonstrate knowledge of the fundamentals of editing various types of science copy 99 Enforce ICMJE authorship guidelines 100 Ensure logic and consistency of manuscripts 101 Demonstrate the ability to assess the quality of papers 102 Ensure papers selected are clinically relevant 103 Ensure papers selected have a clear story-line 104 Demonstrate the ability to select material for its merit, interest to readers, and originality alone 105 Ensure papers selected are suitable to the journal 106 Ensure papers selected for review are meaningful 107 Ensure manuscripts are triaged judiciously (for journals that use such a process) 108 Demonstrate the ability to form preliminary opinions on a submitted manuscript’s relevance 109 Demonstrate the ability to make fast, good decisions about papers 110 Demonstrate the ability to make difficult decisions 111 Demonstrate the ability to exercise excellent judgment 112 Handle manuscripts in the areas of one’s expertise and assist in finding persons qualified to handle papers in those areas outside one’s expertise 113 Engage in and maintain interactions and good relations with media 114 Select, curate, and comment on articles for publication 115 Ensure alterations recommended based on peer reviewers’ comments can be justified 116 Demonstrate experience or familiarity with manuscript tracking software (e.g. Discussion ScholarOne, AllenTrack, PeerTrack, BenchPress) 117 Demonstrate aptitude in using technology (computers, Internet, e-mail, Manuscript Submission Systems) to perform his or her editorial duties) 118 Possess a degree in medical editing or be trained as a journal editor 119 Demonstrate the ability to write editorials 120 Demonstrate working knowledge of the language in which the journal is published 121 Demonstrate skills in speed reading, skim reading, and critical reading 122 Demonstrate an aptitude for reading widely, deeply, and continually 123 Demonstrate experience and/or training in medical journal writing 124 Demonstrate understanding of the parts, purposes, and characteristics of tables, charts, graphs, and images Page 14 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Table 3 Competency-related statements a (Continued) Table 3 Competency related statements (Continued) 125 Demonstrate familiarity with scientific units, numerals, symbols, and nomenclature 126 Demonstrate familiarity with the presentation of data and data presentation problems 127 Demonstrate familiarity with the basic concepts of statistics 128 Demonstrate knowledge of literature reviews 129 Demonstrate familiarity with the principles of scientific investigation 130 Demonstrate familiarity with types of evidence 131 Demonstrate familiarity with scientific referencing 132 Demonstrate familiarity with clinical research design 133 Demonstrate knowledge of types of manuscripts 134 Be working towards a deeper understanding of multiple research epistemologies 135 Assist non-native speakers in dealing with language issues VI. Ethics and integrity 136 Demonstrate knowledge of issues around registration (i.e. Discussion trials, systematic reviews, protocols) 137 Demonstrate knowledge of and adherence to the principles of editorial independence 138 Demonstrate expertise in ensuring the ethical integrity of publications 139 Identify and address allegations of fraud or plagiarism 140 Demonstrate understanding of privacy, confidentiality, and anonymity issues 141 Identify and address issues related to conflicts of interest 142 Identify and address issues related to industry-sponsored research 143 Separate decision-making from commercial considerations 144 Demonstrate knowledge of the ethical approval process for research involving humans and animals 145 Ensure the respect and privacy of patients described in clinical studies 146 Safeguard the rights of study participants and animals 147 Demonstrate understanding of issues related to dual-use research (research with multiple purposes or applications) 148 Identify and apply appropriate reporting guidelines 149 Guarantee access to, and long term preservation of, the published information 150 Encourage debate on important topics related to the journal 151 Promote higher standards of medical journalism 152 Identify and work to avoid publication bias 153 Demonstrate knowledge of COPE resources for editors, authors, and peer reviewers 154 Demonstrate knowledge of copyright issues 155 Demonstrate knowledge regarding problems with multiple publications (e.g. salami, duplicate, redundant) 156 Identify and address incongruities and bias in manuscripts 157 Recommend publication of papers that meet standards of scientific rigor 158 Identify and address issues related to image manipulation VII. Qualities and characteristics of editors 159 Demonstrate experience and broad knowledge of the field(s) covered by the journal and of the people working in thos 160 Demonstrate the ability to work in a team 161 Delegate/divide the workload 162 Communicate clearly with others 163 Effectively summarize manuscripts in fields outside your experience 164 Possess a Doctorate or Master’s Degree in related content area 165 Demonstrate an academic education that includes science training or experience in a research environment 166 Demonstrate experience and aptitude in conflict resolution Page 15 of 17 Galipeau et al. BMC Medicine (2016) 14:16 research team are editors. Discussion Finally, due to the broad in- clusion criteria and the decision to preserve the wording used by authors to describe potential competencies as much as possible, it is lik tually be competencies tasks, behaviors, and kn Table 3 Competency-related statements a (Continued) 167 Demonstrate excellent organizational, project, and time management skills, including the ability pressure 168 Maintain part time professional practice 169 Maintain membership in learned societies and editing-related associations 170 Be recognized as a distinguished scholar in one’s field 171 Maintain an active research portfolio/is employed in a research-oriented university or institute 172 Demonstrate past experience on an editorial board 173 Demonstrates competence as a practitioner in their field 174 Demonstrate strong interpersonal skills 175 Demonstrate good analytical skills 176 Demonstrate effective critical appraisal skills 177 Demonstrate the ability to achieve consensus among opinionated scientists 178 Demonstrate leadership skills 179 Demonstrate political and public relations sense 180 Demonstrate self-motivation 181 Demonstrate enthusiasm 182 Demonstrate tolerance and persistence 183 Demonstrate boldness 184 Demonstrate independent thinking 185 Maintain visibility and respect among peers and in the larger scientific community 186 Maintain rigid criteria 187 Demonstrate the ability to perpetuate or challenge master narratives 188 Exercise convictions with a positive attitude 189 Demonstrate a willingness to reconsider decisions 190 Demonstrate practicality 191 Demonstrate decisiveness 192 Demonstrate personal interest in medical ‘journalology’ or ‘editology’ 193 Demonstrate an enjoyment of learning and a questioning mind 194 Demonstrate the desire to advance their field of study 195 Have access to a good academic network or have the potential to grow one 196 Demonstrate patience when dealing with authors and reviewers 197 Demonstrate knowledge of processes related to the editorial board 198 Respond promptly to complaints 199 Act with integrity and accountability 200 Engage with social media to reach out beyond the usual specialist audiences 201 Demonstrate knowledge of the parts, purposes, and characteristics of manuscripts 202 Demonstrate knowledge of open access models Other potential competencies 203 Statements related specifically to the Editor-in-Chief position d a The order in which the statements are presented is purely for purposes of organization and is not intended to conv c Corresponds to the Item(s) columns in Tables 1 and 2 b Number of extracted competency-related statements across all data sources in the scoping review d This item contains all statements pertaining only to potential competencies of Editors-in-Chief. Discussion Despite these poten to this scoping review, we nevertheless wanted to account for them in our results as they did fit our inclusion criteria 169 Maintain membership in learned societies and editing-related associations 5 170 Be recognized as a distinguished scholar in one’s field 8 171 Maintain an active research portfolio/is employed in a research-oriented university or institute 5 172 Demonstrate past experience on an editorial board 1 173 Demonstrates competence as a practitioner in their field 2 174 Demonstrate strong interpersonal skills 5 175 Demonstrate good analytical skills 6 176 Demonstrate effective critical appraisal skills 4 177 Demonstrate the ability to achieve consensus among opinionated scientists 1 178 Demonstrate leadership skills 20 179 Demonstrate political and public relations sense 3 180 Demonstrate self-motivation 5 181 Demonstrate enthusiasm 4 182 Demonstrate tolerance and persistence 5 183 Demonstrate boldness 6 184 Demonstrate independent thinking 4 185 Maintain visibility and respect among peers and in the larger scientific community 4 186 Maintain rigid criteria 1 187 Demonstrate the ability to perpetuate or challenge master narratives 1 188 Exercise convictions with a positive attitude 1 189 Demonstrate a willingness to reconsider decisions 8 190 Demonstrate practicality 3 191 Demonstrate decisiveness 4 192 Demonstrate personal interest in medical ‘journalology’ or ‘editology’ 3 193 Demonstrate an enjoyment of learning and a questioning mind 3 194 Demonstrate the desire to advance their field of study 15 195 Have access to a good academic network or have the potential to grow one 2 196 Demonstrate patience when dealing with authors and reviewers 4 197 Demonstrate knowledge of processes related to the editorial board 10 198 Respond promptly to complaints 7 199 Act with integrity and accountability 39 200 Engage with social media to reach out beyond the usual specialist audiences 22 201 Demonstrate knowledge of the parts, purposes, and characteristics of manuscripts 3 202 Demonstrate knowledge of open access models 2 Other potential competencies 203 Statements related specifically to the Editor-in-Chief position d 68 a The order in which the statements are presented is purely for purposes of organization and is not intended to convey any type of ranking c Corresponds to the Item(s) columns in Tables 1 and 2 b Number of extracted competency-related statements across all data sources in the scoping review d This item contains all statements pertaining only to potential competencies of Editors-in-Chief. Authors’ contributions DM i d f h d DM conceived of the study, participated in its design and coordination, and helped to draft the manuscript. JG participated in the design of the study, coordinated the research, and was involved in title/abstract and full-text screening, data extraction and verification, and writing of the manuscript. LS was involved in the design of the study, as well as participating in title/abstract and full-text screening, and data extraction and verification. SS was involved in the design of the study. KDC was involved in data extraction and verification. EW, SB, MC, HM, JD, MW, PG, VB, PT, and PB all provided content expertise. All authors were involved in the analysis and interpretation of data, revision of drafts of the manuscript, and reading and approving the final manuscript. Abbreviations COPE: Committee on Publication Ethics; WAME: World Association of Medical Editors. COPE: Committee on Publication Ethics; WAME: World Association of Medical Editors. Conclusion To our knowledge, this scoping review is the first at- tempt to systematically identify possible competencies of editors. On its own, the review will serve to in- form readers on the extent and nature of existing lit- erature in this area, as well as the breadth of skills, abilities, tasks, knowledge, and training that may be necessary to fulfill the position of scientific editor at a biomedical journal. More importantly, the review is part of a larger program to develop a minimum set of core competencies for scientific editors of biomed- ical journals. Acknowledgments W th k C h We thank Cochrane, the Committee on Publication Ethics (COPE), the World Association of Medical Editors (WAME), the European Association of Science Editors (EASE), and the Council of Science Editors (CSE) for their partnership in this scoping review. We thank Becky Skidmore for developing and running the search strategy and Raymond Daniel for acquiring, uploading, and managing records in DSR. We thank Jennifer Tetzlaff (JT) and Mona Ghannad (MG) for their involvement in screening titles and abstracts. This project is funded by Cochrane, Elsevier, and BioMed Central. The purpose of the 203 competency-related state- ments generated here is to be the central tool used for a Delphi exercise involving scientific editors of biomedical journals from around the world. Subsequently, these statements will serve to stimulate discussion at a con- sensus meeting in which the goal will be for relevant stakeholders to agree upon a minimum set of core competencies for scientific editors of biomedical jour- nals. This evidence-based approach will ultimately lay the groundwork for the development of specific competency-based training and certification for scien- tific editors of biomedical journals [5]. The develop- ment of core competencies and subsequent training represent critical steps toward ensuring that the pub- lication of biomedical research truly represents a hall- mark of quality and trustworthiness, both within and beyond the research community. Competing interests DM is supported by a University of Ottawa Research Chair. EW is a self- employed consultant and provides training for editors, authors, and reviewers. She has received payment for such training from a variety of publishers, academic societies, universities, and commercial companies. She was also involved in developing the International Standards for Editors and many of the COPE guidelines. SB, MC, and HM are part of the Cochrane Central Editorial Unit. JD leads the Cochrane Collaboration’s test evaluation activities. PG is a Coordinating Editor for the Cochrane Infectious Diseases Group. MW participated in the creation and dissemination of a survey to WAME members on medical journal editor professionalism in June 2015 and in the development of a series of sessions on medical journal editor professionalism for the WAME International Conference for Medical Journal Editors in New Delhi held in October 2015. JG, SS, LS, KDC, VB, PT, and PB all have no competing interests to declare. Another limitation is that for feasibility purposes we only considered English and French articles, which raises the possibility that relevant information published in another language was missed. Similarly, the databases searched may not have included some journals from out- side fiscally resourced countries. Discussion Despite these potential competencies not being directly relevant to this scoping review, we nevertheless wanted to account for them in our results as they did fit our inclusion criteria 68 much as possible, it is likely that some items may not ac- tually be competencies per se, but may instead describe tasks, behaviors, and knowledge related to competencies. research team are editors. Finally, due to the broad in- clusion criteria and the decision to preserve the wording used by authors to describe potential competencies as much as possible, it is likely that some items may not ac- tually be competencies per se, but may instead describe tasks, behaviors, and knowledge related to competencies. Page 16 of 17 Galipeau et al. BMC Medicine (2016) 14:16 Additional files However, these items are still useful in describing im- portant aspects of editors’ work and will therefore con- tribute valuable information for the development of core competencies. Additional file 1: Search strategies. (DOCX 23 kb) Additional file 2: Non-research-based publications. (DOCX 49 kb) With the large number of competency statements and our desire to create a manageable list for use down- stream in our program of research, our efforts to remove redundant and overlapping items in order to streamline the list may also have led to the elimination of some nu- ances between items that were subtly different from one another. While we implemented measures to ensure consistency in our methods (i.e. piloted forms, duplica- tion of the classification exercise), ultimately there is a degree of interpretation and selectivity embedded in this process. Thus, our list of possible competencies may not include all of the competencies of biomedical editors. As noted above, the next phases of this project are designed to elicit any missing items. Received: 2 December 2015 Accepted: 20 January 2016 References 1. Groves T. 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Accessed 4 Nov 2014. 23. Marusic A, Katavic V, Marusic M. Role of editors and journals in detecting and preventing scientific misconduct: strengths, weaknesses, opportunities, and threats. Med Law. 2007;26(3):545. 23. Marusic A, Katavic V, Marusic M. Role of editors and journals in detecting and preventing scientific misconduct: strengths, weaknesses, opportunities, and threats. Med Law. 2007;26(3):545. Galipeau et al. BMC Medicine (2016) 14:16 Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit and we will help you at every step: • We accept pre-submission inquiries
https://openalex.org/W4205350474	https://ojs.mtak.hu/index.php/fogorv-szemle/article/download/4883/3832	Hungarian	null	Minimál invazív bemeneti kavitás premoláris és moláris fogaknál – irodalmi összefoglalás	Fogorvosi szemle	2,018	cc-by	4,921	A bemeneti kavitás bült gyökércsatornákat [28]. Az EVH azonban elenged hetetlen a nikkel-titánium (NiTi) műszerek alkalmazásá nál is. Annak ellenére, hogy ezek a műszerek nagyon rugalmasak, EVH hiányában torzulhatnak és el is tör hetnek ciklikus fáradás következtében [27, 28]. A gyökérkezelés első lépése a bemeneti kavitás kialakítá sa, amely kulcsfontosságú a kezelés sikeressége szem pontjából. Korrekt gyökérkezelés elvégzése a gyökér csatornák kemo-mechanikai preparálásával valósul meg. Műszereinknek és fertőtlenítő oldatainknak köz vetlen kapcsolatba kell kerülniük a gyökércsatornák falával, ez azonban nem érhető el teljes mértékben a gyökércsatorna-rendszer komplexitása miatt. A ren delkezésre álló preparáló eszközök és átöblítő szerek tulajdonságait figyelembe véve a bemeneti kavitás pre parálásának célja és alapelvei, melyek az egyetemen oktatásra kerülnek [12] A bemeneti kavitás végleges formájának kialakításá hoz a kavitás falait ki kell terjeszteni és simára preparálni, hogy koronai dentin ne akadályozza az EVH-t és a csa torna apikális szakaszának megmunkálhatóságát. Ehhez szükséges a pulpakamra tetejének teljes eltávolítása. A folyamat során alávájások keletkezhetnek a pulpakam ra falán, ép foganyag felesleges eltávolítása mellett [7, 8]. Az EVH további akadálya lehet az orifíciumoknál talál- ható dentinpolc. Ennek eltávolításához javasolták koráb ban a Gates-Glidden- és a Peeso-fúrók használatát, vi szont az orifíciumok túlzott feltágítása a külső dentinfal és a furkáció felőli dentinfal elvékonyodásához, ezáltal a fog töréssel szemben mutatott ellenállásának csökke néséhez vezet [7, 8, 13, 14, 23]. A törési ellenállás nö velésének céljából Clark és Khademi 2010-ben közölte a minimál invazív bemeneti kavitás kialakításának kon cepcióját moláris fogak vonatkozásában [8]. oktatásra kerülnek [12]: – a szuvas foganyag és az elégtelen széli zárású ko ronai restaurátumok teljes eltávolítása [6, 34] – az ép foganyag megőrzése [1, 32, 34] – a pulpakamra tetejének teljes eltávolítása [1, 34] – a pulpaszövet teljes eltávolítása [34] – az orifíciumok lokalizálása [34] – az orifíciumok lokalizálása [34] – egyenes vonalú hozzáférhetőség (EVH) biztosítása a foramen apicale-hoz vagy a csatorna első görbü letéhez [1, 6, 34]. FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. 123–128. n FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. 123–128. n Semmelweis Egyetem – Konzerváló Fogászati Klinika DR. BALÁZS MÓNIKA, DR. KOMORA PÉTER, DR. TÓTH ZSUZSANNA A közösségi médiában és a tudományos fórumokon is egyre többször jelennek meg esetbemutatások, melyekben mini atűr bemeneti kavitás(ok)on keresztül végeznek gyökérkezelést a vállalkozó szellemű fogorvosok. Az egyértelmű techni kai bravúrhoz elengedhetetlen nagyítás (operációs mikroszkóp használata), megfelelő megvilágítás és műszerezettség. Minimál invazív bemeneti kavitás kialakításával a gyökérkezelés kivitelezése nehezebb és időigényesebb lehet, ezért összetettebb anatómiájú fogaknál csak korlátozottan alkalmazható. Egyszerűbb esetekben azonban, ha a szövettörme lék eltávolítása és a pulpaűr fertőtlenítése biztosított, valós alternatívája lehet a hagyományos bemeneti kavitásoknak. Több koronai foganyag megőrzését teszi lehetővé, így a fog restaurálható marad, de a törési ellenállás növekedését in vitro egyelőre nem sikerült igazolni. Kulcsszavak: minimál invazív bemeneti kavitás, pericervikális dentin, törési ellenállás, operációs mikroszkóp Érkezett: 2018. február 5. Elfogadva: 2018. október 30. Minimál invazív bemeneti kavitás premoláris és moláris fogaknál – irodalmi összefoglalás DR. BALÁZS MÓNIKA, DR. KOMORA PÉTER, DR. TÓTH ZSUZSANNA Minimál invazív bemeneti kavitás A fog nyaki régiója kapja a legtöbb terhelést funkció közben [2], ezért itt fordul elő a legtöbb fraktúra [17], ami gyakran – Touré és mtsai. (2011) szerint az esetek 15,1%-ban [33]. – szerepel a gyökérkezelt fogak eltávo lításának indokaként [12, 35]. A fogak törési ellenállása közvetlen összefüggést mutat a megmaradt ép foganyag mennyiségével [6, 13, 17, 32]. Kisőrlőket vizsgálva megállapították, hogy a be meneti kavitás önmagában csak kis hatással (5%) van a fog szilárdságára. Azonban, ha ez az approximális zá- rólécek preparációjával is jár (MOD kavitás), akkor már 63%-os csökkenést figyelhetünk meg [13, 17]. A módszert leíró szerzők nem értenek egyet azzal, hogy minden csatornabemenetnek egyszerre kell lát hatónak lennie a tükörben, mivel ez túlzottan sok dentin eltávolítását teszi szükségessé [1, 23]. A minimál in- vazív bemeneti kavitás kisebb méretéből adódóan kor látozza a látási viszonyokat. Az emberi szem felbontó- képessége kb. 0,2 mm, ami operációs mikroszkóp (OM) használatával 0,6 µm-re növelhető. Így kijelenthető, hogy ez a kavitásforma csak nagyítás és megfelelő megvilágítás használata mellett alakítható ki megbíz hatóan [3, 5, 6, 18, 20, 22, 23, 27, 30, 36]. A dentin – összetételének köszönhetően – rugalmas szövet, törékenysége kisebb, mint a zománcé, akár még alámenősen is megfelelő ellenállással rendelkezik. Clark és Khademi (2010) megfigyelése szerint ezek az aláme nős területek nagy értéket képviselhetnek a fog számá ra. A szerzők szerint a pulpaszarvak felett megmaradó 0,5–3 mm széles dentinperem hasonlóan egy abroncs hoz, belső merevítésként szolgálhat a fog számára [8]. A dentinperem alól a pulpaszarvak ultrahangos eszkö zökkel és átöblítőszerekkel mikroszkópos kontroll alatt teljes mértékben eltávolíthatók, a terület áttisztítható [23]. A kezelést befolyásolja a fog pozíciója a fogívben, dőlése, a szájnyitási korlátozottság, a kalcifikáció mér téke, valamint a kezelőorvos képzettsége, gyakorlottsá ga és technikai felkészültsége. A trepanációs kavitás ki alakításának első fázisa a pulpakamra tisztítása, amely során szükséges az orifíciumokhoz az EVH-t akadá lyozó dentin és zománc eltávolítása. Gyémántbevona tú vagy bevonattal nem rendelkező ultrahangos fejek használatával vízhűtés nélkül is finoman preparálható a pulpakamra alja és falai, így a látási viszonyok is job bak [6] (1. kép). A második fázisban a csatornabeme netek megkereséséhez az egészséges foganyag meg őrzése érdekében mikro-műszereket (pl. micro-openert vagy pilot-reszelőt) és speciális endodonciai fúrókat is alkalmazhatunk [6]. Ezek a hosszú szárú és nyakú, kis fejű acélgömbfúrók, elsősorban mikroszkópos munká hoz (2. kép). Minimál invazív bemeneti kavitás esetén a gyökércsatornák megmunkálásához rugalmas ötvö zetből készült tágítókat kell választani. Minimál invazív bemeneti kavitás NiTi file-ok hasz nálatával a gyökércsatornák eredeti lefutása megőriz hető a preparáció során [6, 18, 30]. j [ ] Gyökértömött fogaknál a koronai zárás döntő fontos ságú, befolyásolja a gyökérkezelés hosszútávú sikeres ségét. Megfelelő abroncshatás biztosítása a nyaki régió ban elengedhetetlen egy jól záró korona készítéséhez. Csapos műcsonk készítéséhez minimum 2 mm magas és minimum 1 mm széles ép foganyag szükséges [8, 17]. Csappal ellátott fog esetében alacsony kockázattal ak kor számolhatunk, ha csak az egyik approximális fal hiányzik. Ha hiányzik a bukkális/orális fal vagy csak a bukkális és orális fal található meg, akkor közepes koc kázattal kell számolnunk a hosszútávú sikeresség szem pontjából [17]. Ezek alapján elmondható, hogy a legjobb megközelítés a gyökérkezelt fogak helyreállíthatósága érdekében az, hogy minimalizáljuk az ép foganyag eltá volítását, különösen a nyaki régióban az abroncshatás megvalósulása érdekében [10, 32]. Minimál invazív bemeneti kavitás A fogak gyökércsatornái többnyire nem egyenes lefu tásúak, különösen jellemző az apikális harmad görbü lete, de egyes fogaknál már a középső harmadban is találkozhatunk különböző fokú görbületekkel. EVH biz tosításával csökkenthető a preparációs hibák előfordu lása [26]. Ezeket főként a vastag, merev acélműszerek okozhatják azáltal, hogy próbálják kiegyenesíteni a gör A minimál invazív bemeneti kavitás alkalmazását támo gató szerzők szerint a hagyományosan kialakított beme- neti kavitás koncepciója alapvetően hibás, mert a ké- nyelmes hozzáférhetőségi forma kialakításával elsősorban a kezelőorvos számára biztosítja a gyökérkezelés elvég DOI https://doi.org/10.33891/FSZ.111.4.123-128 FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n 124 Minimál invazív bemeneti kavitás kialakításához szük séges a fog anatómiájának részletes tanulmányozása. A kavitás tervezéséhez első lépésként a kiindulási rönt- genfelvétel / felvételek vagy a még pontosabb három dimenziós képet adó Cone Beam CT (CBCT)-felvétel gondos elemzésére van szükség. Meg kell figyelni a pe- ricervikális régió anatómiáját, a gyökércsatornák lefutá- sát, a pulpakamra magasságát és alapjának szélességét mesio-distalis, és mélységét vestibulo-oralis irányban. Figyelembe kell venni a szuvasodás, illetve a cserére szoruló restaurátum helyzetét [4, 23]. Mivel a legna gyobb görbülethez biztosított EVH a pericervikális és furkációs dentin károsodásával járhat, az orifíciumok hoz érdemes egyenes vonalú hozzáférhetőséget ter vezni (orifice-oriented access) [11, 23]. Ezzel csator nánként 0,97 ± 0,32 mm vastag pericervikális dentin eltávolítása előzhető meg egy mikro CT- analízissel ké szült vizsgálat szerint [11]. zéséhez szükséges feltételeket [7, 8]. Ezen feltételek biz tosításával azonban gyakran a fog restaurátummal való ellátásának és hosszútávú megtarthatóságának esé- lye csökken. A hagyományos bemeneti kavitás kialakí tásánál javasolt EVH forma kialakítása a pericervikális dentin gyengüléséhez vezet. A pericervikális dentin az alveoláris csontszéltől apikálisan és koronálisan számí tott 4-4 mm foganyagot jelenti [8]. A fognyaknál találha tó dentin vastagsága kulcsfontosságú a fog helyreállít hatósága és a fog szerkezeti integritása szempontjából. A fog nyaki régiója kapja a legtöbb terhelést funkció közben [2], ezért itt fordul elő a legtöbb fraktúra [17], ami gyakran – Touré és mtsai. (2011) szerint az esetek 15,1%-ban [33]. – szerepel a gyökérkezelt fogak eltávo lításának indokaként [12, 35]. zéséhez szükséges feltételeket [7, 8]. Ezen feltételek biz tosításával azonban gyakran a fog restaurátummal való ellátásának és hosszútávú megtarthatóságának esé- lye csökken. A hagyományos bemeneti kavitás kialakí tásánál javasolt EVH forma kialakítása a pericervikális dentin gyengüléséhez vezet. A pericervikális dentin az alveoláris csontszéltől apikálisan és koronálisan számí tott 4-4 mm foganyagot jelenti [8]. A fognyaknál találha tó dentin vastagsága kulcsfontosságú a fog helyreállít hatósága és a fog szerkezeti integritása szempontjából. FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n kép: Endodonciai fúrók csatornakereséshez és dentin szelektív eltávolításához (CJM Engineering – Munce Discovery Burs) [15] 3. kép: Minimál invazív bemeneti kavitások sémás ábrázolása a hagyományossal szemben: A: két különálló bemenet, B: X vagy lépcsős, C: csonkagúla alakú. 3. kép: Minimál invazív bemeneti kavitások sémás ábrázolása a hagyományossal szemben: A: két különálló bemenet, B: X vagy lépcsős, C: csonkagúla alakú. FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n 125 1. kép: Gyémántbevonatú ultrahangos preparáló fejek a pulpakamra falainak minimál invazív alakításához (VDW Diamond-coated CAVI tips) [16]. 2. kép: Endodonciai fúrók csatornakereséshez és dentin szelektív eltávolításához (CJM Engineering – Munce Discovery Burs) [15] 1 kép: Gyémántbevonatú ultrahangos preparáló fejek 1. kép: Gyémántbevonatú ultrahangos preparáló fejek a pulpakamra falainak minimál invazív alakításához (VDW Diamond-coated CAVI tips) [16]. tása megközelítőleg egyenes, lehetőség nyílik két kü lönálló bemenet készítésére a mesialis és distalis gyö kércsatorna-rendszerek számára. Ebben az esetben a pulpakamra tetejének középső része érintetlen marad (truss), ezáltal a fog húzó- és nyomóerőkkel szemben ellenállóbb maradhat [8]. Keskeny pulpakamra alappal és konvergáló vagy erősen görbült gyökércsatornák kal rendelkező fogaknál X alakú, más néven lépcsős bemenet alakítható ki. A kavitás ilyenkor leginkább egy homokórára hasonlít: a pulpakamra alapja felé és occlusalis irányba is széttérő. Széles alap és erő sen konvergáló csatornák, valamint keskeny alapú, de egyenes lefutású csatornák esetén a bemeneti kavitás gyakran egy csonkagúlához hasonlít (3. kép). Lehető ségünk van egy meglévő approximális restaurátumon vagy szuvas, de már kitisztított lézión keresztül, az oc clusalis felszín megnyitása nélkül is elvégezni a gyö kérkezelést, ha az egyenes vonalú hozzáférhetőség az orifíciumokhoz így is biztosítható (1. táblázat). A beme neti kavitás kialakítását kiegészíthetjük a zománcszélek ferdére preparálásával vagy a gyökércsatorna képze letbeli koronális folytatásának megfelelő kis bevágások készítésével. Így jobb rálátás biztosítható, és a tágító műszerek torzulása is elkerülhető. 1. kép: Gyémántbevonatú ultrahangos preparáló fejek a pulpakamra falainak minimál invazív alakításához (VDW Diamond-coated CAVI tips) [16]. 3. kép: Minimál invazív bemeneti kavitások sémás ábrázolása a hagyományossal szemben: A: két különálló bemenet, B: X vagy lépcsős, C: csonkagúla alakú. 2. kép: Endodonciai fúrók csatornakereséshez és dentin szelektív eltávolításához (CJM Engineering – Munce Discovery Burs) [15] 1. kép: Gyémántbevonatú ultrahangos preparáló fejek a pulpakamra falainak minimál invazív alakításához (VDW Diamond-coated CAVI tips) [16]. 1. kép: Gyémántbevonatú ultrahangos preparáló fejek a pulpakamra falainak minimál invazív alakításához (VDW Diamond-coated CAVI tips) [16]. 2. kép: Endodonciai fúrók csatornakereséshez és dentin szelektív eltávolításához (CJM Engineering – Munce Discovery Burs) [15] 1. táblázat A leggyakoribb minimál invazív bemeneti kavitások kialakításának lehetőségei. A pulpakamra alapja A gyökércsatorna lefutása Bemeneti kavitás alakja széles egyenes két különálló bemenet konvergáló és/vagy erősen görbült csonkagúla keskeny egyenes csonkagúla konvergáló és/vagy erősen görbült X vagy lépcsős A leggyakoribb minimál invazív bemeneti kavitások kialakításának lehetőségei. 2. kép: Endodonciai fúrók csatornakereséshez és dentin szelektív eltávolításához (CJM Engineering – Munce Discovery Burs) [15] 2. A minimál invazív bemeneti kavitások morfológiája Minden jól kialakított bemeneti kavitás egyensúlyt ké pez a konzervatív és kényelmi forma között és a le hető legkevesebb ép foganyag feláldozásával biztosítja az ideális hozzáférhetőséget a gyökércsatornákhoz [4]. A minimál invazív bemenetképzés nem feltétlenül je lent extrém kisméretű bemeneti kavitásokat [8, 23]. Ha a pulpakamra alapja széles és a gyökércsatornák lefu FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n Ezek alapján kijelenthető, hogy minimál invazív bemeneti kavitás esetében feltétel, hagyományos be menet esetén pedig rendkívül hasznos az OM haszná lata [30]. Figyelembe kell venni, hogy a felső molárisok mesiobuccalis gyökércsatorna-rendszere kihívást je lenthet a járulékos csatornák lokalizálhatósága és fel tárhatósága miatt. A minimál invazív bemeneti kavitás nem minden esetben alkalmas ezeknél a fogaknál [22]. A i i ál i í b i k i á Cl k é Kh d hatóságát, nem fordult elő műszertörés vagy lépcsőkép ződés a kísérlet során. A tágítást V-Taper2H (SS White Lakewood, USA) rendszerrel végezték [22]. A kutatócso port korábban alsó első molárisokkal is végzett hasonló vizsgálatokat, ahol a teljes csatornát nézve viszont a dis talis csatornában szignifikánsan nagyobb volt a minimál invazív csoportban a preparálatlan felszínek aránya. A kü- lönbséget az apikális gyökéri harmadban találták a leg nagyobbnak [19]. A minimál invazív kezelés – több foganyag megőr zéséből következően – a törési ellenállás növekedését ígéri. Ezzel kapcsolatban több ex vivo vizsgálat is zaj lott, egymásnak helyenként ellentmondó eredménnyel. Restaurátummal ellátott felső molárisoknál például nem volt szignifikáns különbség a töréssel szemben mutatott ellenállásban a hagyományos és a minimál invazív cso- port között [22, 30]. Özyürek és mtsai. (2018) minimál in- vazív (truss) és hagyományos bemenetet alakítottak ki mesio-occlusalis kavitásokból alsó molárisokban. A gyö- kércsatornák tágítása és tömése után a fogat kom pozit restaurátummal látták el. Ebben a vizsgálatban sem volt szignifikáns különbség, a fraktúra minőségét vizsgálva azonban a minimál invazív csoportban szig nifikánsan több restaurálható fogat találtak [26]. Más kísérletben, ahol a fogakat nem látták el semmilyen restaurátummal és üreget csak occlusalisan alakítot tak ki, a premolárisok és molárisok kontroll- és minimál invazív csoportja szignifikánsan ellenállóbbnak mutat kozott, mint a hagyományos csoport. A vizsgálat során megfigyelték, hogy premolárisoknál a fractura a minimál invazív csoportban csak a csücsköket érintette, nem pe- dig a kavitás teljes falát [19]. Plotino és mtsai. (2017) kí sérletüket az ultrakonzervatív „ninja” bemeneti kavitással kibővítve végezték. Direkt restaurátummal ellátott alsó és felső premolárisokat és molárisokat vizsgáltak stati kus terhelésnek kitéve a fogakat. A hagyományos cso port szignifikánsan alacsonyabb törési ellenállást muta tott, mint a kontroll, minimál invazív és „ninja” csoportok. A „ninja” bemenet nem növelte a törési ellenállást a mi nimál invazívhoz képest. Itt is vizsgálták a fraktúra minő ségét. Az összes preparált csoportban a nem helyreállít ható törések száma volt szignifikánsan magasabb a nem preparált kontrollcsoporthoz képest, az egyes csoportok között azonban nem volt szignifikáns eltérés [29]. FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n 126 lönbség a lupéval vagy OM segítségével végzett ke zelések között [5]. Egy másik vizsgálatban, ahol fog orvostan-hallgatók végezték a vizsgálatokat, OM se gítségével szignifikánsan nagyobb arányban észlelték a gyökércsatorna-bemeneteket, mint lupéval vagy sza bad szemmel [36]. Minimál invazív bemeneti kavitások vonatkozásában eddig csak egy in vitro tanulmány ké szült. Szabad szemmel, OM segítségével, valamint OM és ultrahangos preparáló eszközök segítségével vé gezték a vizsgálatot felső első moláris fogakon. Minimál invazív bemeneti kavitás esetén szignifikánsan keve sebb MB2 gyökércsatorna feltárása volt lehetséges OM segítségével, de OM és ultrahangos preparálás együt tes alkalmazásával már közel azonos eredményt értek el [30]. Ezek alapján kijelenthető, hogy minimál invazív bemeneti kavitás esetében feltétel, hagyományos be menet esetén pedig rendkívül hasznos az OM haszná lata [30]. Figyelembe kell venni, hogy a felső molárisok mesiobuccalis gyökércsatorna-rendszere kihívást je lenthet a járulékos csatornák lokalizálhatósága és fel tárhatósága miatt. A minimál invazív bemeneti kavitás nem minden esetben alkalmas ezeknél a fogaknál [22]. A minimál invazív bemeneti kavitás Clark és Khade- mi (2010) által megfogalmazott célja a pericervikális den tin védelme és megőrzése [8]. Krishan és mtsai. (2014) felső nagymetsző, alsó második premoláris és alsó el- ső moláris fogakon vizsgálta az eltávolított dentin men� nyiségét hagyományos és minimál invazív bemene tek esetén. A vizsgálat során kiderült, hogy a módszer minden fogcsoportban kevesebb dentin eltávolításával járt összességében, de szignifikáns különbség csak a koronai részben volt, a gyökércsatorna koronális egyharmadában nem [19]. Egy másik vizsgálatban megfigyelték, hogy minimál invazív bemenet esetén a tágítás akaratlanul a kavitás megnagyobbodásához vezetett gépi forgó műszerek használatával. A keze lőorvosnak figyelembe kell vennie a bemenetképzés nél azt, hogy milyen méretű legyen a kavitás ahhoz, hogy a gépi tágítókban keletkező felesleges stresszt elkerülje [25]. A következő vizsgálatokban a gyökércsatornák meg lönbség a lupéval vagy OM segítségével végzett ke zelések között [5]. Egy másik vizsgálatban, ahol fog orvostan-hallgatók végezték a vizsgálatokat, OM se gítségével szignifikánsan nagyobb arányban észlelték a gyökércsatorna-bemeneteket, mint lupéval vagy sza bad szemmel [36]. Minimál invazív bemeneti kavitások vonatkozásában eddig csak egy in vitro tanulmány ké szült. Szabad szemmel, OM segítségével, valamint OM és ultrahangos preparáló eszközök segítségével vé gezték a vizsgálatot felső első moláris fogakon. Minimál invazív bemeneti kavitás esetén szignifikánsan keve sebb MB2 gyökércsatorna feltárása volt lehetséges OM segítségével, de OM és ultrahangos preparálás együt tes alkalmazásával már közel azonos eredményt értek el [30]. A minimál invazív bemeneti kavitás értékelése az irodalom alapján Megfelelő bemeneti kavitás hiányában nehézségbe üt közik a pulpaűr teljes feltárása. Nem maradhat a mik roorganizmusok szaporodásához rejtett zug, mint pél dául egy feltáratlanul maradt pulpaszarv a pulpakamra tetejének részleges eltávolítása esetén [27, 29]. Neela kantan és mtsai. (2018) a pulpakamrában és a mesialis gyökércsatornákban maradt szövettörmelék mennyisé gét vizsgálták hagyományos és két különálló / truss bemeneti kavitás esetén. A gyökércsatornákban nem találtak különbséget, a pulpakamrában azonban szig nifikánsan több szövettörmelék maradt minimál invazív bemeneti kavitás esetén [24]. A csak részlegesen feltárt pulpakamra ezen túl növeli az esélyét annak, hogy feltáratlan gyökércsatorna marad a kezelt fogak ban [21, 29]. 3. kép: Minimál invazív bemeneti kavitások sémás ábrázolása a hagyományossal szemben: 3. kép: Minimál invazív bemeneti kavitások sémás ábrázolása a hagyományossal szemben: A: két különálló bemenet, B: X vagy lépcsős, C: csonkagúla alakú. tornák lokalizálhatóságában már több vizsgálatban is bizonyították. Az egyik tanulmány azt mutatja, hogy na gyítás alkalmazásával – akár lupe, akár operációs mik roszkóp (OM) – felső moláris fogakban a mesiobucca- lis 2 (MB2) csatorna észlelése háromszor sikeresebb volt, mint nagyítás nélkül, de nem volt szignifikáns kü- A nagyítás használatának előnyét a járulékos csa FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n Irodalom 1. Adams N, Tomson PL: Access cavity preparation. Br Dent J. 2014: 333–339. Bóveda és Kishen (2015) javasolják a CBCT rutin szerű használatát minimál invazív bemenetképzés ter vezésénél [3]. Connert és mtsai. (2014) szerint azon ban nincs szignifikáns különbség az eltávolított dentin mennyiségében a CBCT és a periapikális röntgenfel vétel alapján tervezett bemeneti kavitások esetében, így a CBCT-felvétel ilyen szempontból nem jelentett előnyt a tervezésnél [9]. Az azonban kétségtelen, hogy a CBCT nyújtotta nagy felbontású, részletgazdag felvé telen jól értékelhetők a fog egyedi anatómiai jellemzői. Zehnder és mtsai. (2016) pre- és postoperatív CBCT segítségével értékelték az in vitro 3 dimenzióban digi tálisan tervezett, irányított gyökérkezeléseket („guided endodontics”), és azt találták, hogy a nyomtatott sablon használatával pontos bemeneti kavitást és a gyökér csatornák apikális harmadig való jó hozzáférhetőségét lehet biztosítani [37]. 2. Asundi A, Kishen A: A strain gauge and photoelastic analysis of in vivo strain and in vitro stress distribution in human dental sup porting structures. Arch Oral Biol. 2000: 543–550. 3. Bóveda C, Kishen A: Contracted endodontic cavities: the founda tion for less invasive alternatives in the management of apical periodontitis. Endodontic Topics. 2015: 169–186. 4. Buchanan LS: Cutting endodontic access cavities – for long-term outcomes. Roots. 2015: 60–63. 5. Buhrley LJ, Barrows MJ, BeGole EA, Wenckus CS: Effect of magnification on locating the MB2 canal in maxillary molars. J Endod. 2002: 324–327. 6. Bürklein S, Schafer E: Minimally invasive endodontics. Quintes sence Int. 2015: 119–124. 7. Clark D, Khademi JA: Case Studies in Modern Molar Endodon tic Access and Directed Dentin Conservation. Dent Clin N Am. 2010: 275–289. 8. Clark D, Khademi JA: Modern Molar Endodontic Access and Di rected Dentin Conservation. Dent Clin N Am. 2010: 249–273. 9. Connert T, Rein D, ElAyouti A, Godt A: Does CBCT help to re duce the amount of dentine removal during access cavity prepa ration? – abstract. Int Endod J. 2014: 50–113. Az eddigi vizsgálatokon túl további ex vivo vizsgála tokra van szükség, hogy a gyökércsatorna-tágítás, -tisz- títás és -fertőtlenítés minőségét meg tudjuk ítélni. Ezt követően randomizált esetkontrollos vizsgálatok, vala mint retrospektív és prospektív vizsgálatok is szüksé gesek, melyek eredményeinek függvényében a meg felelő indikációkban megfontolható a minimál invazív bemeneti kavitás kialakításának rutinszerű alkalma- zása [31]. 10. Összefoglalás 13. Gluskin AH, Peters C, Peters OA: Minimally invasive endodon tics: challanging and prevailing paradigms. Br Dent J. 2014: 347– 353. Minimál invazív bemenetképzés esetén a pulpakamrában több szövettörmelék marad, ezért ennek eltávolításá hoz operációs mikroszkóp és ultrahangos preparáló fe jek használata javasolt. Ezen eszközök alkalmazása to vábbá javítja az összes gyökércsatorna megtalálásának esélyét, ahogyan kiindulási CBCT-felvétel készítése is, amely a bemeneti kavitás tervezésében is segítségünk re lehet. A minimál invazív módszer pericervikális den tinre gyakorolt védő hatását nem sikerült még kimutatni, viszont a fog koronai részében egyértelműen több fog anyag marad, ami a fog restaurálhatóságát nézve nem utolsó szempont. A tágító műszerek erős deformációja ellenére a megmunkálás hatékonyságában és sikeressé gében nincs különbség, bár a gyökércsatornák áthelye ződésével számolni kell. Egyelőre nincs bizonyíték arra vonatkozóan, hogy a minimál invazív módszerrel növel hető lenne a fog törési ellenállása. Ö 14. Gutmann JL: Minimally invasive dentistry (Endodontics). J Cons Dent. 2013: 282–283. 15. http://www.dentalproductshopper.com/munce-discovery-burs 16. https://www.vdw-dental.com/en/products/detail/cavi-tips/ 17. Jotkowitz A, Samet N: Rethinking ferrule – a new approach to an old dilemma. Br Dent J 2010: 25–33. 18. Khademi JA, Trudeau M, Narayana P, Rabi RM, Baerg SD: Image- guided endodontics: the role of the endodontic triad https://www. dentalcetoday.com/courses/261%2FPDF%2FDT_August_16_ NPR_Khademi.pdf (2016.10.24.) 19. Krishan R, Paqué F, Ossareh A, Kishen A, Dao T, Friedman S: Im pacts of conservative endodontic cavity on root canal instrumen tation efficacy and resistance to fracture assessed in incisors, premolars, and molars. J Endod. 2014: 1160–1166. 20. Mamoun JS: The maxillary molar endodontic access opening: A microscope-based approach. Eur J Dent. 2016: 439–446. 21. Mathew ST, Rajan JS: Minimally invasive endodontics. J Dent Oral Hyg. 2014: 36–38. 22. Moore B, Verdelis K, Kishen A, Dao T, Friedman S: Impacts of Contracted Endodonic Cavities on Instrumentation Efficacy and Biomechanical Responses in Maxillary Molars. J Endod. 2016: 1779–1783. Összefoglalásképpen elmondható, hogy a gyökér kezelés megbízhatóan kivitelezhető megfelelő tárgyi és személyi feltételek esetén minimál invazív beme neti kavitáson keresztül is, de egyelőre még nem si került in vitro vizsgálatok alapján teljes mértékben igazolni a módszer előnyeit, illetve egyetlen klinikai vizsgálat sem készült a kezelés sikerességének vo natkozásában. 23. Narayana P: Access Cavity Preparations. In Schwartz RS, Canakapalli V (ed.): Best Practices in Endodontics: A Desk Ref erence. Quintessence Publishing, USA, 2015; 89–103. 24. FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n A be meneti kavitás restaurátummal való ellátásának módja a fogak töréssel szemben mutatott ellenállását nagy ban befolyásolja. Ezért továbbra is a csücsköket vagy a teljes fogat borító definitív restaurátum készítése java solt posterior fogak esetében [22]. Az eddigi vizsgálato kat összesítő szisztematikus áttekintés alapján egyelő re nincs tudományos bizonyíték, amely alátámasztaná, hogy a minimál invazív bemeneti kavitás a hagyomá nyossal szemben szignifikánsan növelné a gyökérkezelt fogak törési ellenállását [31]. Niemi és mtsai. (2016) megemlítették, hogy a vizsgá A minimál invazív bemeneti kavitás Clark és Khade- mi (2010) által megfogalmazott célja a pericervikális den tin védelme és megőrzése [8]. Krishan és mtsai. (2014) felső nagymetsző, alsó második premoláris és alsó el- ső moláris fogakon vizsgálta az eltávolított dentin men� nyiségét hagyományos és minimál invazív bemene tek esetén. A vizsgálat során kiderült, hogy a módszer minden fogcsoportban kevesebb dentin eltávolításával járt összességében, de szignifikáns különbség csak a koronai részben volt, a gyökércsatorna koronális egyharmadában nem [19]. Egy másik vizsgálatban megfigyelték, hogy minimál invazív bemenet esetén a tágítás akaratlanul a kavitás megnagyobbodásához vezetett gépi forgó műszerek használatával. A keze lőorvosnak figyelembe kell vennie a bemenetképzés nél azt, hogy milyen méretű legyen a kavitás ahhoz, hogy a gépi tágítókban keletkező felesleges stresszt elkerülje [25]. A következő vizsgálatokban a gyökércsatornák meg munkálásának hatékonyságát vizsgálták. Rover és mtsai. (2017) felső első molárisokat vizsgáltak, a tágítás hoz Reciproc (VDW München, Németország) rendszert használtak. A preparálatlan gyökércsatorna-felszínek ará nyában nem volt szignifikáns különbség, ahogyan a fel halmozódott dentintörmelék mennyiségében sem a mi nimál invazív és a hagyományos bemeneti kavitáson keresztül végzett tágítás után. A vizsgálat során azon ban azt észlelték, hogy a distobuccalis és palatinalis csatornák lefutása részben megváltozott, a csatornák áthelyeződtek [30]. A Moore és mtsai. (2016) által felső molárisokon végzett vizsgálatban is hasonló eredményre jutottak. A megmunkálás hatékonysága összességében a minimál invazív és a hagyományos bemeneti kavitás esetében is alacsony volt, viszont nem volt szignifikáns különbség a két csoport között. A minimál invazív beme neti kavitás nem veszélyeztette a csatorna megmunkál Niemi és mtsai. (2016) megemlítették, hogy a vizsgá latukban felhasznált alsó kisőrlő fogak esetében a mi- nimál invazív bemeneti kavitás nem tette lehetővé a continous wave technikával való gyökértömés elké FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n 127 szítését. Moláris fogaknál nagyobb az esély arra, hogy ezzel a technikával elegendő hely áll rendelkezésre a gyökértömés kivitelezéséhez [25]. Irodalom Dietschi D, Duc O, Krejci I, Sadan A: Biomechanical considera tions for the restoration of endodontically treated teeth: a sys tematic review of the literature, Part II (Evaluation of fatigue be havior, interfaces, and in vivo studies). Quintessence Int. 2008: 117–129. 11. Eaton JA, Clement DJ, Lloyd A, Marchesan MA: Micro–Computed Tomographic Evaluation of the Influence of Root Canal System Landmarks on Access Outline Forms and Canal Curvatures in Mandibular Molars. J Endod. 2015: 1888–1891. Á 12. Fazekas Á: A pulpaűr anatómiája, bemeneti cavitas preparálása és a gyökércsatorna hosszának meghatározása. In Fazekas Á (szerk.): Megtartó fogászat és endodoncia. Semmelweis Kiadó, Budapest, 2006; 287–297. Összefoglalás Neelakantan P, Khan K, Hei Ng GP, Yip CY, Zhang C, Pan Cheung GS: Does the orifice-directed dentin conservation access design debride pulp chamber and mesial root canal systems of man FOGORVOSI SZEMLE 111. évf. 4. sz. 2018. n 128 dibular molars similar to a traditional access design? J Endod. 2018: 274–279. 31. Silva EJNL, Rover G, Belladonna FG, De-Deus G, da Silveira Teixeira C, da Silva Fidalgo TK: Impact of contracted endodon tic cavities on fracture resistance of endodontically treated teeth: a systematic review of in vitro studies. Clin Oral Invest. 2018: 109–118. 25. Niemi TK, Marchesan MA, Lloyd A, Seltzer RJ: Effect of Instru ment Design and Access Outlines on the Removal of Root Ca nal Obturation Materials in Oval-shaped Canals. J Endod. 2016: 15150–1554. 32. Tang W, Wu Y, Smales RJ: Identifying and reducing risks for po tential fractures in endodontically treated teeth. J Endod. 2010: 609–617. 26. Özyürek T, Ülker Ö, Demiryürek EÖ, Yilmaz F: The effects of endodontic access cavity preparation design on the fracture strength of endodontically treated teeth: traditional versus con servative preparation. J Endod. 2018: 800–805. 33. Touré B, Faye B, Kane AW, Lo CM, Niang B, Boucher Y: Analysis of reasons for extraction of endodontically treated teeth: a pro spective study. J Endod. 2011: 1512–1515. servative preparation. J Endod. 2018: 800–805. 27. Patel S, Rhodes J: A practical guide to endodontic access cavity preparation in molar teeth. Br Dent J. 2007: 133–140. 34. Vertucci FJ, Haddix JE: Tooth Morphology and Access Cavity Preparation. In Hargreaves KM, Cohen S: Cohen’s Pathways of the Pulp. (10th ed.) Elsevier, St. Loius, 2010; 136–222. 28. Peters OA: Current challenges and concepts in the preparation of root canal systems: a review. J Endod. 2004: 559–567. 35. Vire DE: Failure of endodontically treated teeth: classification and evaluation. J Endod. 1991: 338–342. 29. Plotino G, Grande NM, Isufi A, Ioppolo P, Pedulla E, Bedini R et al.: Fracture strength of endodontically treated teeth with different access cavity designs. J Endod. 2017: 995–1000. 36. Yoshioka T, Kobayashi C, Suda H: Detection rate of root canal ori fices with a microscope. J Endod. 2002: 452–453. 30. Rover G, Belladonna FG, Bortoluzzi EA, De-Deus G, Silva EJNL, Teixeira CS: Influence of access cavity design on root canal de tection, instrumentation efficacy, and fracture resistance assessed in maxillary molars. J Endod. 2017: 1657–1662. 37. Összefoglalás Zehnder MS, Connert T, Weiger R, Krastl G, Kühl S: Guided en dodontics: accuracy of a novel method for guided access cavity preparation and root canal location. Int Endod J. 2016: 966–972. Balázs M, Komora P, Tóth Zs Keywords: minimally invasive access cavity, pericervical dentin, orifice oriented access, fracture resistance, operating microscope Minimally invasive access cavity in premolars and molars – review There is an increasing number of case presentations in the social media and scientific forums, in which dentists and endodontists perform successful root canal treatments through tiny access cavities. This technical achievement cannot be carried out without proper instruments, magnification (operating microscope) and lighting. With a minimally invasive access cavity the root canal treatment itself can be complicated and time-consuming. In case of a tooth with complex anatomy the use of this method is limited. On the other hand, in simpler cases, if debris is eliminated and the pulp cavity is disinfected properly, it can be a real alternative for traditional access cavities. It enables us to preserve more coronal tooth structure, which makes final restoration easier to do, but the increase in fracture resistance has not been proved yet in vitro. Keywords: minimally invasive access cavity, pericervical dentin, orifice oriented access, fracture resistance, operating microscope Pályázat Körmöczi-pályadíjra A pályázat beadási határideje: 2019. február 4. A pályázatokat, kérem, emailen juttassák el címemre! A pályázat beadási határideje: 2019. február 4. A pályázatokat, kérem, emailen juttassák el címemre! Dr. Fejérdy Pál az MFE főtitkára fejerdy.pal@dent.semmelweis-univ.hu Felhívjuk minden, a Fogorvosi Szemlében publikáló, rendezett MFE tagsággal rendelkező, 35 évnél fiata labb első szerzős cikk szerzőjét, hogy pályázzanak a 2018-as Körmöczi-pályadíjra. Felhívjuk minden, a Fogorvosi Szemlében publikáló, rendezett MFE tagsággal rendelkező, 35 évnél fiata labb első szerzős cikk szerzőjét, hogy pályázzanak a 2018-as Körmöczi-pályadíjra. A pályázatokat, kérem, emailen juttassák el címemre! Dr. Fejérdy Pál az MFE főtitkára fejerdy.pal@dent.semmelweis-univ.hu Pályázni csak a 2018-ban a Fogorvosi Szemlében megjelent közleményekkel lehet. Kérjük, a közlemény pdf változatát mellékelje a pályázathoz. Pályázni csak a 2018-ban a Fogorvosi Szemlében megjelent közleményekkel lehet. Kérjük, a közlemény pdf változatát mellékelje a pályázathoz.
https://openalex.org/W3213915247	https://journal.lifescifi.com/index.php/RH/article/download/17/13	English	null	Database Management System in Accounting: Assessing the Role of Internet Service Communication of Accounting System Information	Research Horizon	2,021	cc-by-sa	2,154	Research Horizon Vol. 1, no. 3, (2021), 100-105 Website: http://journal.publindoakademika.com/index.php/RH Database Management System in Accounting: Assessing the Role of Internet Service Communication of Accounting System Information M. Fakhimuddin1,, Uswatun Khasanah2, Rini Trimiyati3 M. Fakhimuddin1,, Uswatun Khasanah2, Rini Trimiyati3 1,2,3 Universitas Islam Negeri Sunan Kalijaga Yogyakarta * Corresponding author: Email: ibnusulaiman996@gmail.com Received : Feb 08 2021 Revised : May 09, 2021 Accepted : Jun 02, 2021 1,2,3 Universitas Islam Negeri Sunan Kalijaga Yogyakarta 1,2,3 Universitas Islam Negeri Sunan Kalijaga Yogyakarta Keywords Received : Feb 08 2021 Revised : May 09, 2021 Accepted : Jun 02, 2021 database management system, accounting, internet service communication, accounting system information Abstract Data management is part of information resource management which includes all activities that ensure that company data resources are accurate, up-to-date, and safe from tampering, and are also available to users and the company. This study aims to find out about the role of concept database management in managing large volumes of company data. The result showed that data management activities include data collection, integrity, and testing, storage, maintenance, security, organization, retrieval. Meanwhile, the database structure includes hierarchical database structure, network database structure, and relational database structure. E- ISSN 2807-9531 P-ISSN 2808-0696 E- ISSN 2807-9531 P-ISSN 2808-0696 1. Introduction In the current era of globalization, management information systems are an inseparable part of an organization where information systems produce output using inputs to meet the needs of achieving management goals (McLeod & Schell, 2007). Database management systems organize the large volumes of data that a company uses in its day-to-day transactions. Data must be organized so that managers can find specific data easily and quickly to make decisions. Companies break down the entire data collection into a set of interconnected data tables, these M. Fakhimuddin, Uswatun Khasanah, Rini Trimiyati small collections of interconnected data will reduce data repetition so that in the end the consistency and accuracy of the data eats up (Samson & Aponso, 2020) Before the database era, companies experienced limitations in their data management because of the way data was organized in secondary storage. Initial attempts to overcome this obstacle included sorting and merging files/documents, extensive computer programming to search and match document records, and document indexes and links built into the data records. The database concept is built on indexes and links to achieve a logical relationship between multiple documents (Lightstone et al., 2010). Most companies use databases that follow a relational structure. Two important reasons behind using this structure are that relational database structures are easy to use and the relationships between tables within the structure are implicit. The ease of use has emboldened many managers to become direct users and sources of databases (Paredaens et al., 2012). This study aims to find out about the role of concept database management in managing large volumes of company data. 101 \| R e s e a r c h H o r i z o n 2. Database Management and Hierarchy From this arrangement, archives/documents are the highest level, while data elements are the lowest level (Gaol, 2008). Then the traditional data hierarchy is archives/documents (files), note (record) and data element. From this arrangement, archives/documents are the highest level, while data elements are the lowest level (Gaol, 2008). Because data is a resource, and this process is called data management. Data management is part of information resource management which includes all activities that ensure that company data resources are accurate, up-to-date, and safe from tampering, and are also available to users. Data management activities include: 1. Data collection. The required data is collected and recorded in a form, which is called a source document which functions as input for the system. 2. Integrity and testing. The data is checked to ensure consistency and accuracy based on predetermined rules and constraints. 3. Storage. Data is stored on a medium, such as magnetic tape or magnetic disk. 4. Maintenance. New data is added, existing data is properly modified, and data that is no longer needed is deleted to keep data resources up-to-date. 5. Security. Data is safeguarded to prevent destruction, damage or misuse. 6. Organization/ The data is structured in such a way as to meet the user's information needs. 7. Taking. Data is available to users. Before the computer era, all activities were carried out by administrative staff supported by punched cards and machine-driven machines which were still primitive in nature. Today, people are still needed for a lot of data collection and testing, but computers have taken up most of the data management responsibilities (Gaol, 2008). 2. Database Management and Hierarchy Data management is part of information resource management and ensures that a company's data resources accurately reflect the physical systems they represent. Data resources are stored in secondary/second storage, which can be either sequential or direct access. Magnetic tape is the most popular sequential storage medium, and magnetic disks are the primary means of achieving direct access. However, the new direct access technology, the compact disk, is gaining popularity today (Ahmad et al., 2003). The software that manages the database is called a database management system (DBMS) (Dayal & Goodman, 1982) . All DBMSs have a data description language processor (DDL processor) which is used to create the database, as well as a database manager that provides the database to users. Users using data manipulation and query languages are high-level language software or software languages that resemble human language, which allows users to easily extract data and information from a database. The person in charge of the database and DBMS is the database administrator or DBA for short (Van Aken et al., 2017) Most of the attention is now directed to very large databases, called database warehouses. The retrieval process, called data mining, provides a higher level of support for the user than usual. DBMS provides a real advantage for companies that use computers as an information system (Gaol, 2008). Companies have traditionally organized their data in a hierarchy, which consists of data, records and document (Onder & Sundus, 2013). The data element (data element) is the smallest data unit, which cannot be further divided into meaningful units. In the salary record, the data elements are: name, employee number, social security number, hourly wages, and number of dependents. A higher level of the hierarchy are records. A record consists of all data elements related to a particular object or activity. For example, there are records that describe each type of inventory and sales. All similar records are organized into one document. Document is a collection of data records (data records) associated with a particular object. For example, an Open Purchase Order that describes a purchase order that has been ordered to a primary supplier, but has not yet been received. 101 \| R e s e a r c h H o r i z o n Database Management System in Accounting: Assessing…… Then the traditional data hierarchy is archives/documents (files), note (record) and data element. 3. Conceptual Framework of Direct-Access Storage Device (DASD) A database is a collection of computer data that is integrated, organized, and stored in a way that facilitates retrieval (Solihin et al., 2017). DASD should be used. Figure 1 shows that multiple company records can be logically integrated. This logical integration of records in multiple archives is called the database concept. The lines in the drawing represent logical integration. Vol. 1, no. 3 (2021), 100-105 \| 102 M. Fakhimuddin, Uswatun Khasanah, Rini Trimiyati , , y Figure 1. Direct-Access Storage Device of Company Records Salesperson files Sales statistics file Customer files Accounts receivable file Buyer files Investment files Supplier files Accounts payable file Buyer order file General ledger file Sales statistics file Supplier files Buyer files General ledger file Buyer order file Figure 1. Direct-Access Storage Device of Company Records The two main objectives of the database concept are minimizing data redundancy and achieving data independence. Data repetition is data duplication, meaning that the same data is stored in several archives. This repetition of data can also be called pleonasm. Data independence is the ability to make changes in the data structure without making changes to the program that processes the data. Data independence is achieved by placing data specifications in tables and dictionaries that are physically separate from the program. Programs refer to tables to access data. Changes to the data structure are only made once, namely in the table. When companies adopt the database concept, the data hierarchy becomes a database (database), archives (files), records (records), data elements (data elements) (Gaol, 2008). 103 \| R e s e a r c h H o r i z o n 4. Conclusion Data management is part of information resource management and ensures that a company's data resources accurately reflect the physical systems they represent. The software that manages the database is called a database management system (DBMS). All DBMSs have a data description language processor (DDL processor) which is used to create the database, as well as Database Management System in Accounting: Assessing…… a database manager that provides the database to users. Companies have traditionally organized their data in a hierarchy, which consists of elements, records, and files. Data management activities include: data collection, integrity and testing, storage, maintenance, security, organization, retrieval. A database is a collection of computer data that is integrated, organized, and stored in a way that facilitates retrieval. The two main objectives of the database concept are minimizing data redundancy and achieving data independence. Database structure is the way data is organized for efficient data processing. The database structure includes hierarchical database structure, network database structure, and relational database structure. The database was created based on several steps, namely: determining data requirements, explaining the required data, and entering data into the data. There are several important personnel related to the database. The database administrator has both technical and managerial responsibility for database resources. Database programmers are required to code efficient data processing computers. Database end users are another important database personnel. Through the decisions they make and the amount of data taken, end users have a huge impact on database design, use and efficiency. Solihin, W., Eastman, C., Lee, Y. C., & Yang, D. H. (2017). A simplified relational database schema for transformation of BIM data into a query-efficient and spatially enabled database. Automation in Construction, 84, 367-383. Van Aken, D., Pavlo, A., Gordon, G. J., & Zhang, B. (2017, May). Automatic database management system tuning through large-scale machine learning. In Proceedings of the 2017 ACM International Conference on Management of Data (pp. 1009-1024). 105 \| R e s e a r c h H o r i z o n 105 \| R e s e a r c h H o r i z o n References Ahmad, A., Ramakrishnan, A., McLean, M. A., & Breau, A. P. (2003). Use of surface plasmon resonance biosensor technology as a possible alternative to detect differences in binding of enantiomeric drug compounds to immobilized albumins. Biosensors and Bioelectronics, 18(4), 399-404. Choi, M. J., Lim, J. J., Torralba, A., & Willsky, A. S. (2010, June). Exploiting hierarchical context on a large database of object categories. In 2010 IEEE computer society conference on computer vision and pattern recognition (pp. 129-136). IEEE. Dayal, U., & Goodman, N. (1982, June). Query optimization for CODASYL database systems. In Proceedings of the 1982 ACM SIGMOD international conference on Management of data (pp. 138-150). , ( ) f j ; p Lightstone, S. S., Teorey, T. J., & Nadeau, T. (2010). Physical Database Design: the database professional's guide to exploiting indexes, views, storage, and more. Morgan Kaufmann. McLeod, R., & Schell, G. P. (2007). Management information systems (Vol. 10). Upper Saddle River New Jersey 07458: Pearson/Prentice Hall. Onder, E., & Sundus, D. A. G. (2013). Combining analytical hierarchy process and TOPSIS approaches for supplier selection in a cable company. Journal of Business Economics and Finance, 2(2), 56-74. Paredaens, J., De Bra, P., Gyssens, M., & Van Gucht, D. (2012). The structure of the relational database model (Vol. 17). Springer Science & Business Media. Raymond, B., & Hosie, G. (2009). Network-based exploration and visualisation of ecological data. Ecological Modelling, 220(5), 673-683. Samson, S., & Aponso, A. (2020, May). An Analysis on Automatic Performance Optimization in Database Management Systems. In 2020 World Conference on Computing and Communication Technologies (WCCCT) (pp. 6-9). IEEE. Vol. 1, no. 3 (2021), 100-105 \| 104 M. Fakhimuddin, Uswatun Khasanah, Rini Trimiyati Solihin, W., Eastman, C., Lee, Y. C., & Yang, D. H. (2017). A simplified relational database schema for transformation of BIM data into a query-efficient and spatially enabled database. Automation in Construction, 84, 367-383. Van Aken, D., Pavlo, A., Gordon, G. J., & Zhang, B. (2017, May). Automatic database management system tuning through large-scale machine learning. In Proceedings of the 2017 ACM International Conference on Management of Data (pp. 1009-1024). 105 \| R e s e a r c h H o r i z o n
https://openalex.org/W4391326667	https://www.degruyter.com/document/doi/10.1515/opth-2022-0244/pdf	English	null	“… God Said”: Toward a Quantum Theology of Creation	Open Theology	2,024	cc-by	14,051	Research Article https://doi.org/10.1515/opth-2022-0244 received October 06, 2023; accepted January 11, 2024 Abstract: This work argues that because Quantum Mechanics (QM) is superb at simulating natural, physical phenomena, so-called quantum-native phenomena, and that Quantum Natural Language Processing (QNLP), its child, is also quantum-native, God’s generative language of physical creation is native to a quantum world; therefore, it is now possible to postulate an argument for God’s creation of the world through language without requiring divine revelation. This argument is, in essence, an extension of the Augustinian argument – the baseline argument from mathematics and abstract objects – for the existence of God that accounts for con- temporary advances in QM. Additionally, this work invites further research and investigation into applying category theory to model the theistic universe thereby allowing both modeling and simulations of the symbolic world. Keywords: quantum computing, theology, QNLP, category theory, DisCoCat, Genesis, John, creation, cos- mology, symbolism  * Corresponding author: Ryder Dale Walton, Capitol Technology University, Doctoral Programs, Laurel, Maryland, United States, e-mail: rdwalton@captechu.edu, rdnotlaw91@gmail.com, rdnotlaw91@outlook.com Open Theology 2024; 10: 20220244 Open Access. © 2024 the author(s), published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0 International License. Keywords: quantum computing, theology, QNLP, category theory, DisCoCat, Genesis, John, creation, cos- mology, symbolism 1.1 Purpose In the search for truth, the present work considers creation in light of new advances in QM and computer science (CS). The intersection of these two disciplines is often known as quantum computing (QC). The signature boon that QC provides over classical computing is performing and solving problems related to large-scale simulation of natural, physical phenomena.1 Such problems are called quantum-native.2 It is no stretch of the imagination that simulation of particles using computers built with particles would be quantum-native. There is a much more surprising quantum-native problem domain known as quantum natural language processing (QNLP) that is the focus of the present work. QNLP is the quantum version of natural language processing (NLP), which is responsible for technologies such as text-to-speech, speech-to-text, autocorrect, auto-complete, personal assistants, AI chatbots, Chat-GPT, and more. NLP on classical computers has brought an immense number of advancements in technology using machine learning techniques and statistics over large corpora of texts. These techniques are often conglomerated into a single concept known as the bag-of- words approach.3 In essence, this means that classical computers are excellent at guessing the next words by performing sophisticated mathematics on a stored vector of word counts. QNLP is diﬀerent because quantum computers can know grammar and syntax because they have suﬃcient computational space to compute grammar and syntax.4 This is precisely what is so surprising about QNLP: that language is quantum-native. It is certainly unexpected that QNLP is quantum-native since it implies a mathematical connection between language and physical reality. We ﬁnd this same connection expressed in the creative language of God in the Abrahamic faiths. In particular, the Hebrew Bible chronicles that God speaks, and the cosmos – structurally and materially – comes into being.5 When God speaks words, he speaks elementary particles. In a world where God creates with speech, grammar is analogous to the laws of physics. In that analogy, the words are matter and energy,6 and the building-blocks of language, then, are the building-blocks of reality. Figure 1 presents this observation graphically. It all starts with the elementary particles, such as electrons and quarks, which would be nothing more than a pixel in Figure 1. The action really starts with the 36 marks.7 The sub-atomic marks assemble into atomic letters. The atomic letters then combine into compounds called words. than ever before. Therefore, to honor the Logos, it is worth revisiting what was known and juxtaposing it with what now can be seen in nature via QM in hopes of discovering a richer, clearer conception of the truth. than ever before. Therefore, to honor the Logos, it is worth revisiting what was known and juxtaposing it with what now can be seen in nature via QM in hopes of discovering a richer, clearer conception of the truth. than ever before. Therefore, to honor the Logos, it is worth revisiting what was known and juxtaposing it with what now can be seen in nature via QM in hopes of discovering a richer, clearer conception of the truth.  1 Feynman, “Simulating Physics;” Aaronson, Quantum. 2 Coecke et al., “Foundations.” 3 Ibid.; Coecke, “Text Structure.” 4 Coecke, “Text Structure.” 5 New Revised Standard Version, Genesis 1-2:3. 6 Because it is now known that mass and energy are interconvertible via special relativity’s = E mc2 equation, then the analogy between words also extends to energy. 7 The number 36 is derived teleologically, meaning that the number is determined by the end of the ﬁgure. The sentence “There was light” requires 36 marks in these shapes to be constructed. 8 Hebrews 11:3. 1 And Now, We See More There have been things to see – things worth seeing – longer than there have been eyes, but ever since the dawn of vision the perplexing problem of making sense out of what is seen has plagued the eyes, and the minds, of the beholders. It is a trivial matter to see the fact of a lion eating a lamb, or that the rain falls on everyone when it comes. It is an entirely diﬀerent matter to see into the events and understand their under- lying mechanisms and causes – how they work. Still further beyond this is the bottomless depth of what these things mean. One man sees a ﬂying bird and knows it means good luck. A woman, instead, sees the same bird and knows the change of the season will soon be upon her. What is more, it is not as if the observable world – the seen – has remained static through the course of time. A contemporary mind riddled with paranoia might observe the ﬂying bird and fear with a deep sense of dread that it is a camera, which means that the observer may actually be the observed and that the world may be more Orwellian than it feared. While that may sound ludicrous, in the contemporary world, robotic birds and cameras, at the very least, exist. Perhaps, the idea is not so ludicrous after all. Stranger still, inﬁnitesimally small particles, invisible to the naked eye, are visible with the help of advanced optical tooling and computers. When one accounts for the passage of time, the state of the contemporary Cartesian mind – the zeitgeist – of the beholders, and the state of the various arts and sciences, it is no wonder that, for many, the search for meaning is fruitless or altogether abandoned. Yet, this lurking despair and gradual retreat into nihilism is far from inevitable. The secret to seeing well is to remain ﬁrmly based on observable reality via science and the time-tested tradition encapsulated in the Logos simultaneously. Advances in contemporary physics, quantum mechanics (QM) in particular, and com- puter technology have made it possible to see more of the world – have made more of the world observable – 2  Ryder Dale Walton 2 Figure 1: Building blocks of language: the metaphysical analogue to a physical world. This ﬁgure comes from Pageau’s Language of Creation, which discusses the cosmology and symbolism of the ancient mind as expressed in Genesis. It is used with the author’s permission. 1.2 Contributions and Avenues for Further Research In agreement with that thesis, this speculative enterprise provides two novel contributions. First, it presents and explores an initial formulation of an argument for creation via speech without necessarily relying on divine revelation. Stated explicitly, there is an argument for the existence of QNLP in a creation narrative akin to Genesis 1-2 or John 1, though not necessarily those accounts precisely, that relies solely on observing quantum computing experiments. This argument, then, abductively implies that theism is a possible explana- tion for the quantum-native nature of language and that further development of a quantum theistic inter- pretive framework is an avenue for further research toward which this endeavor merely points. Additionally, as a necessary consequence of the ﬁrst contribution, a second novel contribution presents an initial step toward a category theory-based representation of theistic cosmology – a universe created out of heaven-stuﬀ and earth-stuﬀ.9 This initial step can be expanded to fuller representations that account for speciﬁc theological nuances, such as sacraments and sacramentals, eﬀects of sin or baptism on the soul, and so on, in future works. 9 Augustine, Confessions. 1.1 Purpose Finally, the word-compounds are arranged according to grammatical law to form viable sentences capable of expressing an ordered cosmology. The parallel between this vision of language and that of the physical world is perfect. Therefore, while it once seemed that the Abrahamic idea that creation through speech is obscure and discernible through divine revelation alone, in the quantum era where language and physical interactions can be visualized pictorially, such a notion ought to be revisited, examined, and updated to align with the observable world as it can be seen now. Certainly, the creation of the world through the speech of God will always require faith, but there is no need for that faith to be blind given contemporary tools.8  1 Feynman, “Simulating Physics;” Aaronson, Quantum. 2 Coecke et al., “Foundations.” 3 Ibid.; Coecke, “Text Structure.” 4 Coecke, “Text Structure.” 5 New Revised Standard Version, Genesis 1-2:3. 6 Because it is now known that mass and energy are interconvertible via special relativity’s = E mc2 equation, then the analogy between words also extends to energy. 7 The number 36 is derived teleologically, meaning that the number is determined by the end of the ﬁgure. The sentence “There was light” requires 36 marks in these shapes to be constructed. 8 Hebrews 11:3. “… God Said”: Toward a Quantum Theology of Creation  3 2.1.1 Deﬁned The ﬁrst noteworthy background discipline is category theory, which is the branch of mathematics concerned with maximizing abstraction by seeking unifying formal structures so that ﬁndings apply to the broadest possible range of disciplines and problems. Category theory is key because it provides a foundational math- ematical and abstract unity between physics and grammar. This is achieved by creating a deﬁnition of a category and then applying diﬀerent rules and stipulations to the said category until it adequately represents observable phenomena or correctly describes the behavior of a system. Due to their ﬂexibility, there are a wide variety of categories.10 Only the most basic type must be considered here. All categories are constructed of objects and morphisms.11 Objects look like dots or circles and are the nouns of a category. They can be thought of the things inside the category. Morphisms, on the other hand, look like arrows and are the verbs – the transformations an object can undergo to, for example, change it into a diﬀerent object – within the category. Mathematically speaking, morphisms have a domain and a co-domain, which are objects within the category. Simply put, the domain is the input – like a vertex in a graph – into a morphism, and the co-domain is the corresponding output – another vertex.12 Aside from objects and morph- isms, it is crucial that categories allow composition, meaning that morphisms within a category can be nested.13 This nesting of morphisms is roughly equivalent to function composition in algebra in that the nested function is calculated ﬁrst with its output being provided as the input into the next function – i.e., f g x ( ( )). In category theory notation, the most basic composition equivalent to the prior example is typically represented as ∘ f g – read as “f after g.”14 This composition is required to be associative – the order of computation is irrelevant – and there must be an identity morphism that maps an object onto itself – a vertex with an arrow circling back on itself.15 Formally, associativity is deﬁned by the following equation: ∘ ∘ f g h ( ) = ∘ ∘ f g h ( ), and identity is deﬁned as f = ∘ f Iw = ∘ Ix f . Figure 2 provides a visual aid with a graph and labels to aid understanding. 4  Ryder Dale Walton 4  Ryder Dale Walton 2.1.1 Deﬁned While it is possible to add additional rules and restrictions – more structure – to categories, only objects, morphisms, and compositionality with associativity and identity are required; therefore, those elements are the basic deﬁnition of a category. The core of Figure 2 is the graph with its four vertices – objects – and seven edges – morphisms. The edges are more correctly described as three directed edges, which are morphisms between objects, and four loops that represent the identity morphisms. The object W is the starting point of the graph’s ﬂow. The morphisms f, g, and h move one along the graph toward its terminal point – object Z. The identity morphisms are, again, represented by loops, which circle back onto the vertices from which they originate. This means the origin vertex and terminus vertex are the same for each identity morphism. Thus, the identity morphisms are labeled with an I that has a unique subscript to specify the object to which it is the identity. For example, Iw is the identity morphism that originates from and terminates on object W. The rest of the image is layered and uses brackets to illustrate the symbolic mathematical expressions’ referents. For example, the associativity equation – the topmost equation – states that the path from W to Z is the same path regardless of whether ∘ f g or ∘ g f is calculated ﬁrst.  10 Coecke and Paquette, “Categories.” 11 Ibid.; van de Wetering, “ZX-Calculus.” 12 de Felice, “Categorical Tools.” 13 Coecke and Kissinger, Picturing Quantum. 14 Ibid. 15 Coecke and Paquette, “Categories;” van de Wetering, “ZX-Calculus.” 2 Background The journey from QNLP to a proper cosmogony is clearly a long and interdisciplinary one. Before embarking, it is necessary to review the most relevant background knowledge that makes an observation as bizarre as the creation of the cosmos by a higher power’s metaphysical tongue even possible in the ﬁrst place. This knowl- edge includes diverse disciplines such as category theory, mathematical linguistics, quantum computing, theology, and philosophy. A full representation of all these topics would require a bulky tome ﬁlled with pages of technical writing and lofty jargon. For this exploration, brief introductions and only the brightest highlights must suﬃce. First, the physical disciplines are explored before proceeding to the metaphysical disciplines. 15 Coecke and Paquette, “Categories;” van de Wetering, “ZX-Calculus.” 2.1.2 Category of Creation The highly abstract nature of category theory allows it to represent many diﬀerent things, including the theistic universe. The theistic universe is based on ancient cosmology and contains two types of substances: “… God Said”: Toward a Quantum Theology of Creation  5 Figure 2: Example category theory graph and compositional equations. Figure 2: Example category theory graph and compositional equations. heaven-stuﬀand earth-stuﬀ.16 The heaven-stuﬀmakes up the spiritual aspect of life, such as souls, wisdom, the breath of life, consciousness, angels, demons, and so forth. Heaven-stuﬀprovides meaning, hierarchical causality, and life among other things. On the other hand, earth-stuﬀprovides the material aspects of life, like ﬂesh, metals, dirt, water, ﬁre, and air. Any categorical representation of the theistic universe must have objects that represent these two distinct kinds. Beyond these two objects, a wider diversity of possible representations begins to emerge depending on one’s particular theological preferences. In particular, the theistic universe has a third object to describe where heaven-stuﬀand earth-stuﬀmeet: symbol.17 Symbols, then, are the third kind that holds the intersection of heaven-stuﬀand earth-stuﬀ. In Christian theology, this would include human beings themselves as they are made from earth-stuﬀand ﬁlled with the breath of God.18 In sacramental theologies that recognize transubstantiation, this would also include, among other things, the Eucharistic Host. Figure 3 shows a pictorial depiction of the theistic universe that will serve as the starting point for building a notional categorical representation. Figure 3 comes from Pageau’s 2018 work.19 The left-hand side of the image shows the meeting of heaven- stuﬀand earth-stuﬀin Adam, which is elaborated below. The heaven-stuﬀand earth-stuﬀare depicted in semicircles that, when combined, form a whole circle that represents all of creation. Intuitively, heaven would be the upper portion of the circle, and earth would be the lower portion. Next, inward-facing directed edges are used throughout Pageau’s work to graphically depict the formation of symbols – the items depicted in the center – out of their heavenly and earthly elements. A second important observation is shown on the right- hand side. The theistic universe is multi-tiered and recursively depicts the same cosmic structure at diﬀerent levels of granularity, which are called macrocosms and microcosms. This is represented in the ﬁgure by zooming into the center of the macrocosm circle to see the microcosm as if looking through a microscope. The current level of granularity being discussed can be discerned by the topmost directed edge.  16 Augustine, Confessions; Pageau, Language of Creation. 17 Pageau, Language of Creation. 18 Genesis 2:7. 19 Figure 3 is used with Pageau’s permission. 17 Pageau, Language of Creation. 2.1.2 Category of Creation On the left, the granularity is that of the divine. On the right, it is that of humanity. Figure 4 takes the next step, providing a simple abstraction of the theistic universe as a proto-category – a nascent, potential category lacking full deﬁnition – with three objects and morphisms between them. Before viewing the ﬁgure, the morphisms require deﬁnition. There are four in total: I, R, E, and S. Borrowing from computer science, each of these morphisms has been assigned a meaningful name with the single character designations functioning as helpful shorthand. I stands for “informs;” R for “returns;” E for “embodies;” and ﬁnally, S stands for “supports.” The rationale behind the names is simple enough to ascertain. Beginning from the lower right-hand portion of the image and proceeding counterclockwise, the earth supports the symbol. It provides the material element that gives the symbol form, supporting the meaning in a weighty, tangible vessel. The symbol, then embodies the ideal – the wisdom, the breath – of heaven. This also implies, third, that heaven is informing the symbol with a higher principle or meaning; otherwise, the symbol would have nothing to embody. Lastly, now on the bottom-left, the symbol can return to the earth. This would signify the departure of heaven – the desacralization or desecration – from the symbol, rendering the symbol nothing more than an empty vessel – an object devoid of heaven-stuﬀ. Figure 3: Recursive structure of the theistic universe. 6  Ryder Dale Walton 6  Ryder Dale Walton Figure 3: Recursive structure of the theistic universe. y Figure 3: Recursive structure of the theistic universe. designations functioning as helpful shorthand. I stands for “informs;” R for “returns;” E for “embodies;” and ﬁnally, S stands for “supports.” The rationale behind the names is simple enough to ascertain. Beginning from the lower right-hand portion of the image and proceeding counterclockwise, the earth supports the symbol. It provides the material element that gives the symbol form, supporting the meaning in a weighty, tangible vessel. The symbol, then embodies the ideal – the wisdom, the breath – of heaven. This also implies, third, that heaven is informing the symbol with a higher principle or meaning; otherwise, the symbol would have nothing to embody. Lastly, now on the bottom-left, the symbol can return to the earth. 7 “… God Said”: Toward a Quantum Theology of Creation  7 Figure 4 is nothing more than the conceptual starting point for the formation of a category proper, hence the use of the term proto-category. It is worth noting that the four morphisms break down into two sets and that identity morphisms are not yet considered here. There are only those morphisms that pair heaven with the symbol and those that pair earth with the symbol. These two sets can be envisioned as two circular cycles: heaven-symbol and earth-symbol. Envisioning these groups this way can be helpful when considering math- ematical representations of these abstract concepts. For example, it is possible to represent each cycle with an isomorphic invertible function so that the heaven-symbol and earth-symbol sets use the same function. Implementation details like this are simply notional as fully deﬁning the optimal categorical representation of the theistic universe is beyond the scope of this introduction to the concept; however, it is important to recognize that this sort of representation can be formalized mathematically. Notice that the word “humanity” is also included in the symbol object’s circle. This is not an accident. It is the prototypical example! Human beings, as represented by Adam in Figure 2, are the primordial20 blend of heaven- stuﬀ– breath of life – and earth-stuﬀ– dust – because humans are a unity of body and soul.21 Adam is formed out of the dust of the earth and the breath of God from heaven enters Adam’s nostrils. He is, therefore and clearly, a symbol – the combination of heaven and earth – in the category of the theistic universe. Humanity, then, through Adam is the symbol of the unity of heaven with earth more broadly. So, when living righteously, the human being is properly functioning in the theistic universe as the embodiment of heaven on earth. When living wickedly, the human being fails to properly instantiate heaven as a symbol and, therefore, sins. It follows then that the very structure of the theistic universe deﬁnes sin as the botched embodiment of heaven on the earth. The human being’s physical and spiritual lifecycle illumines how the morphisms move the human between the objects in the category. At the moment of death, the breath leaves the body and returns to heaven. The breath, then, no longer informs the meaning of the body. 2.1.2 Category of Creation This would signify the departure of heaven – the desacralization or desecration – from the symbol, rendering the symbol nothing more than an empty vessel – an object devoid of heaven-stuﬀ. Figure 4: Proto-category for the theistic universe. Figure 4: Proto-category for the theistic universe. Figure 4: Proto-category for the theistic universe. 20 Prior to the Fall, Adam and Eve lived in a state of Original Justice that pre-dates Original Sin. During this time, the adjective “perfect” could be ascribed to the symbol of humanity. See Paul II’s Theology of the Body for more thoughts on this topic. 21 Genesis 2:7; Aquinas, Summa, part 1, question 76, article 1. 22 2 S l 12 23 24 Coecke and Paquette, “Categories;” Coecke and Kissinger, Picturing Quantum. 23 Genesis 3:19.  20 Prior to the Fall, Adam and Eve lived in a state of Original Justice that pre-dates Original Sin. During this time, the adjective “perfect” could be ascribed to the symbol of humanity. See Paul II’s Theology of the Body for more thoughts on this topic. 21 Genesis 2:7; Aquinas, Summa, part 1, question 76, article 1. 22 2 Samuel 12:23. 23 Genesis 3:19. The inputs into this process are the proper objects H – heaven- Figure 5: Prototype morphism mapping for a sacramental theology. Figure 6: Converting proto-category into process theoretic representation. 8  Ryder Dale Walton 8  Ryder Dale Walton Figure 5: Prototype morphism mapping for a sacramental theology. 8  Ryder Dale Walton Figure 5: Prototype morphism mapping for a sacramental theology. 8 Ryder Dale Walton Figure 5: Prototype morphism mapping for a sacramental theology. process – a proper morphism – called sacralize. The inputs into this process are the proper objects H – heaven- stuﬀ– and E – earth-stuﬀ. Here, notice that the structure, both physical and logical, of heaven-stuﬀand earth- stuﬀis preserved. Heaven-stuﬀis above the earth. However, it is crucial to realize that connectivity is the key to interpreting these diagrams. Even though E is written in the place of earth-stuﬀ, it connects to the process box on the top, meaning it is an input and not an output. Therefore, attention must be paid to the wires and their connection points rather than the relative positions of the labels. The inputs into – the domain of – the sacralize morphism, then, are the objects H and E. The output – the codomain – is S, meaning a symbol. The same logic is used to create the diagram for the desecrate process, which collapses the notions of the S and R proto-morphisms into a proper morphism. In this case, the symbol S is the domain, and the codomain is H and E. Again, the logical location of H’s label is preserved in this image, but that is not necessary. Figure 5: Prototype morphism mapping for a sacramental theology. Figure 6: Converting proto-category into process theoretic representation. Figure 6: Converting proto-category into process theoretic representation. Figure 6: Converting proto-category into process theoretic representation. process – a proper morphism – called sacralize. The inputs into this process are the proper objects H – heaven- stuﬀ– and E – earth-stuﬀ. Here, notice that the structure, both physical and logical, of heaven-stuﬀand earth- stuﬀis preserved. Heaven-stuﬀis above the earth. However, it is crucial to realize that connectivity is the key to interpreting these diagrams. Even though E is written in the place of earth-stuﬀ, it connects to the process box on the top, meaning it is an input and not an output. This is the great tragedy of death. The soul of the loved one is now gone back to its Maker, and the bereaved have nothing left to do but await their own turns to join their loved one’s soul in heaven as well.22 Meanwhile, the soulless body slowly turns back to dust, returning to the earth, after burial.23 Each of these categorical changes can be mapped as a morphism from the domain of symbol to the co-domain of earth or heaven, respectively. Figure 5 provides a notional, graphical example of such a mapping from a sacramental theology’s perspective. A full exploration of domains and codomains must be left to a future work. There are certainly multiple ways to implement this mapping, so this is only a notional example to aid comprehension. The arrows are precisely those morphisms that are indicated in Figure 4. Additionally, BBSD is an abbreviation meaning “Body, Blood, Soul, and Divinity,” which is the view of transubstantiation regarding the Eucharist. There are other theological positions, but the underlying framework represented here still functions regardless of theological preference. Sacramental theologies tend to have more symbols to consider. With the logical considerations for how the theistic universe is constructed now prototyped, it is time to formalize those considerations into a category proper. The ﬁrst step to doing this is to convert the logical proto- category of Figure 4 into a process theoretic representation. Such a representation automatically provides a formalized mathematical structure that lends itself to implementation on a quantum computer.24 This includes composition structured with associativity and identity. Figure 6 shows the necessary conversion. Figure 4 here is split into two sections representing cycles of heaven-stuﬀto symbol – top – and earth-stuﬀ to symbol – bottom. These sections are then juxtaposed with the equivalent process diagram. This is done to illustrate the conversion to process theory in steps. The two processes will be composed again in Figure 7. The convention employed here is for the processes to be read from top to bottom; therefore, time is labeled with an arrow pointing downward to remove any ambiguity as to the direction of the ﬂow. In that same spirit, the top conversion will be considered ﬁrst. The abstract morphisms I and E have been collapsed into a single process – a proper morphism – called sacralize. Therefore, the composition is associative in process diagrams representative of category theory by default. Identity is shown for objects in the same manner as time. For an object, it is simply an arrow that does not go through a process. The identity morphism is diﬀerent. It is not depicted here because it is simply an empty diagram. A process that makes no change to objects has no need to be represented. Therefore, the composition here perfectly meets the deﬁnition of a category. For ease of understanding the objects, morphisms, domains, and codomains are labeled in this ﬁgure. Domain and codomain are from the perspective of time in this example. So, the inputs are always entering the top of the process box, and outputs are always ﬂowing out of the bottom. Objects are always represented by wires, and morphisms are always represented by boxes and are synonymous with processes. In Figure 7, the labels for objects are no longer bound by the logical structure of Figure 4. That structure can be simpliﬁed because the bends in the wire can be straightened out. Again, so long as the connection point is preserved, only connectivity matters. The image here is now a formalized graph of a category-theoretic process and inherits all the structure and beneﬁts implied. Lastly, it remains to consider which underlying implementation could be used to concretize this category. There is not one simple answer as more than one category is a candidate. The optimal match or best candidate is an area for further research as that is beyond the scope of this contribution, which aims only to invite further research into the intersection of category theory and theology. Therefore, a simple implementation is oﬀered here. Namely, the category of relations known as Rel, which is deﬁned rigorously in Coecke and Paquette and used often to model conceptual spaces among other things.25 Using Rel, the objects in Figure 7 become mathematical sets. The morphisms become mathematical relations, which map sets onto sets. The composition is deﬁned by two relations that relate three sets preserving the same structure found in Figure 2. Therefore, attention must be paid to the wires and their connection points rather than the relative positions of the labels. The inputs into – the domain of – the sacralize morphism, then, are the objects H and E. The output – the codomain – is S, meaning a symbol. The same logic is used to create the diagram for the desecrate process, which collapses the notions of the S and R proto-morphisms into a proper morphism. In this case, the symbol S is the domain, and the codomain is H and E. Again, the logical location of H’s label is preserved in this image, but that is not necessary. process – a proper morphism – called sacralize. The inputs into this process are the proper objects H – heaven- stuﬀ– and E – earth-stuﬀ. Here, notice that the structure, both physical and logical, of heaven-stuﬀand earth- stuﬀis preserved. Heaven-stuﬀis above the earth. However, it is crucial to realize that connectivity is the key to interpreting these diagrams. Even though E is written in the place of earth-stuﬀ, it connects to the process box on the top, meaning it is an input and not an output. Therefore, attention must be paid to the wires and their connection points rather than the relative positions of the labels. The inputs into – the domain of – the sacralize morphism, then, are the objects H and E. The output – the codomain – is S, meaning a symbol. The same logic is used to create the diagram for the desecrate process, which collapses the notions of the S and R proto-morphisms into a proper morphism. In this case, the symbol S is the domain, and the codomain is H and E. Again, the logical location of H’s label is preserved in this image, but that is not necessary. “… God Said”: Toward a Quantum Theology of Creation  9 Figure 7: Composition of the theistic universe as a process diagram. Figure 7: Composition of the theistic universe as a process diagram. Next, the composition is demonstrated in Figure 7. Associativity is included in the composition without needing any diagrammatic amendments or annotations. This is because the top portion of the diagram or the bottom portion of the diagram is calculated ﬁrst. As long as both portions are composed and calculated, the result is the same and the image will look the same. 25 Coecke and Paquette, “Categories;” Heﬀord et al., “Semantic Concepts.” 26 Coecke and Paquette, “Categories.” 10  Ryder Dale Walton 10  Ryder Dale Walton 10 Part of Speech (Po5) The partial ordering here is not of grave importance since the example to follow is simple enough. Should this alphabet be expanded to account for diﬀerences in sentence tense, etc., additional could be required. In such a case, the partial orders ought to be speciﬁed. a tuple from them. This structure is a good starting point since it mandates that symbols be constituted by both heaven-stuﬀand earth-stuﬀ, which ﬁts the abstract proto-categorical model of Figure 4. Further, the use of integers for values allows for a gradation of sacredness – positive values – and profanity – negative values. 0, obviously, would reﬂect neutrality, which would at the very least be useful as an initial value. Such a gradation makes sense as it could model diﬀerences in sanctity between marks on the soul – baptism, ordination, consecration, priests, bishops, and the like – and the relative sacredness of religious objects – bibles, crosses, holy water, rosaries, prayer ropes, and so on. Rel, then, seems like a good starting point for the implementation of the theistic universe as conceived here and in Pageau’s work.27 Now, it may seem like this discussion is a rabbit hole, but it is certainly not. The categorical understanding of the theistic universe postulated here is the spiritual twin of the physical world, and necessarily follows from the main argument of this article as it is a requirement of QNLP that such a unifying, abstract, and mathe- matical structure exists. Therefore, this portion points toward and lays the foundation for the category theoretical understanding of spiritual realities and, by extension, abstract objects – the ﬁrst contribution of this work. It is already documented that category theory can be used for philosophical considerations.28 It is time to apply it theologically, develop it, and put it to the test.  27 Pageau, Language of Creation. 28 Lawvere, “Unity and Identity.” 29 Lambek, “Type Grammar;” de Felice, “Categorical Tools.” 30 Lambek, “Type Grammar;” Cardinal, “Japanese Causatives.” 27 Pageau, Language of Creation. Formally, if there is a relation → R X Y : 1 and a second relation → R Y Z : 2 , then the composition ∘ ⊆ × ≔ ∈ R R X Z x z y Y x R y y R z , there exists a such that and 2 1 1 2 {( )\| } holds.26 Eﬀectively, this is saying that when two relations are composed using the Cartesian product – represented by the multiplication sign – the left-hand side of the relation is populated by an element of the leftmost set, and the right-hand side of the relation is populated by an element of the rightmost set. Again, this structure is exactly that found in the composition deﬁned in Figure 2. Since these properties all hold, then Rel provides a feasible implementa- tion of the theistic universe where the category of earth-stuﬀis deﬁned as ≔ ∈ E e e E , 0 {( ) \| }; heaven-stuﬀis deﬁned as ≔ ∈ H h h H 0, {( ) \| }; symbol is deﬁned as ≔ ∈ ∈ S e h e E h H , , {( ) \| }. Symbol, then, is the Cartesian product of earth-stuﬀand heaven-stuﬀ, which takes e from earth-stuﬀand the h from heaven stuﬀand creates Table 1: The minimal type alphabet for English Type Part of Speech (Po5) n Noun s Sentence The partial ordering here is not of grave importance since the example to follow is simple enough. Should this alphabet be expanded to account for diﬀerences in sentence tense, etc., additional could be required. In such a case, the partial orders ought to be speciﬁed. 10  Ryder Dale Walton 28 Lawvere, “Unity and Identity.” 30 Lambek, “Type Grammar;” Cardinal, “Japanese Causatives.” 2.2 Mathematical Linguistics The diagramed sentence is “John gave Mary a ﬂower.”31 only two nouns and one verb and is still grammatically complete. Figure 8 shows a slightly more complicated example to demonstrate both the simplicity and ﬂexibility of the n type. The diagramed sentence is “John gave Mary a ﬂower.”31 The sentence in question is shown above the type groupings. Each grouping relies on whitespace to associate the types with the word that they represent. Notice also that the n type is also capable of representing adjectives by carrying the nnl encoding. Oftentimes, article adjectives are simpliﬁed out of the calculation before generating a quantum circuit. This minimizes the number of qubits needed, which is necessary on noisy intermediate scale quantum (NISQ) computers because these computers are limited to low qubit counts and incapable of error correction.32 The fact that QNLP is possible on NISQ devices is itself proof of the quantum- native nature of language. Figure 8 warrants some explanation. The pairing of types with adjoints via underwires eﬀectively cancels them out of the calculation. Whatever type remains is yielded on the right side of the arrow. If the yielded type is s, in this simple example, then the collection of words is a grammatically correct sentence. 2.2 Mathematical Linguistics The second background principle of importance is mathematical linguistics, which is necessary to unpack as it formalizes grammar as mathematics. As the name implies, mathematical linguistics is a ﬁeld of research and inquiry where mathematical methods are applied to linguistic phenomena. The ﬁeld is broad, and there exist numerous approaches to represent natural language as mathematics; therefore, the discussion here must necessarily be limited to pregroup grammar, one of the most common approaches used in contemporary QNLP research. Pregroups are a particular kind of category called a monoid that is partially ordered.29 This, in essence, means that there is an order to the relationship of the objects akin to an inequality – the order only ﬂows in one direction. Additionally, for each type, n for example, that is a member of the pregroup P, there are left-adjoints, nl, and right-adjoints, nr, so that expansion and contraction multiplications are possible. As the names imply, expansion is breaking a 1 into its constituent parts. Contraction is combining those same parts, reducing them to a 1. These inequalities are deﬁned as → → n n nn 1 l l and → → nn n n 1 r r , where the arrow is equivalent to a less than or equal to inequality (≤).30 Before continuing, the pregroup P must have an explicitly deﬁned alphabet, or dictionary, of types. Table 1 provides the minimum alphabet necessary to render a grammatical sentence in the English language. Since the minimal alphabet shown in Table 1 contains only n and s, it is simple enough to comprehend. This is perhaps unsurprising since English contains sentences as simple as “Peter jumped up,” which contains “… God Said”: Toward a Quantum Theology of Creation  11 11 only two nouns and one verb and is still grammatically complete. Figure 8 shows a slightly more complicated example to demonstrate both the simplicity and ﬂexibility of the n type. The diagramed sentence is “John gave Mary a ﬂower.”31 The sentence in question is shown above the type groupings. Each grouping relies on whitespace to Figure 8: Pregroup grammar with underwires. Figure 8: Pregroup grammar with underwires. Figure 8: Pregroup grammar with underwires. Figure 8: Pregroup grammar with underwires. only two nouns and one verb and is still grammatically complete. Figure 8 shows a slightly more complicated example to demonstrate both the simplicity and ﬂexibility of the n type. , q 32 Coecke et al., “Foundations;” Coecke et al., “Qubits Speak.” 33 Coecke, “Text Structure.” 34 Coecke and Kissinger, Picturing Quantum. 35 Ibid. 36 DisCoCat is a portmanteau of three words: Categorical Compositional Distributional. It is a rather clever portmanteau since the order of reading composition is why the terms are reversed (think Cat ∘Co ∘Dis). 37 Coecke et al., “Foundations;” Coecke, “Text Structure.” 36 DisCoCat is a portmanteau of three words: Categorical Compositional Distributional. It is a rather clever portmanteau since the order of reading composition is why the terms are reversed (think Cat ∘Co ∘Dis). 37 Coecke et al., “Foundations;” Coecke, “Text Structure.” Figure 9: A DisCoCat diagram. First, this image comes from Karsaklis et al.38 Second, DisCoCat earned its name because these diagrams were developed by using category theory to unify distributional semantics – the linguistics and statistical research approach asserting that words used in the same contexts have similar meanings – and composition into one graphical tool.39 Notice how Figure 9 looks nearly identical to Figure 8. The primary diﬀerences are two-fold: First, the words are written in pentagons; and second, the underwires are curved. Both diﬀerences are signiﬁcant. The pentagons mark a departure from algebraic grammar in as much as they represent processes like those in Figure 7 instead of Figure 8’s types. Regarding the latter diﬀerence, the wires in the diagram are no longer underwires that pair up types with their adjoints. Instead, they are cups. Cups and caps, a cup that is mirrored across the horizontal axis, produce the identity when they are composed, which is the same principle – one of the snake equations – used to transform the processes of Figure 6 into those of Figure 7.40 The use of a cup allows for the output processes to mimic underwires, which reduces the number of intermediary steps and allows for the eventual emergence of simpler quantum circuits.41 Cups and caps, then, are very powerful graphical calculation tools. Having reviewed the mathematic and scientiﬁc background up to the creation of DisCoCat diagrams, there remains only to convert DisCoCat diagrams into quantum circuits using software tools like lambeq and discopy.42 That last step enters the heart of the argument, so it is reserved for the main body of this work. First, there are some theological and philosophical loose ends to tie. 2.4 Theology and Philosophy Theology and philosophy provide the second half of the necessary background information for the present argument. Of course, theology and philosophy are large ﬁelds with millennia of history to consider; therefore, a full treatment on this topic is best left for a much larger work. The present discussion will graft QNLP to the metaphysical tradition by reﬂecting on necessary presuppositions that prepare the mind and the eye to see creation in the circuits. Theology and philosophy will agree that realism and the Logos are indispensable for this preparation, though they will come to terms in their own unique ways.  38 Kartsaklis et al., “Lambeq.” 39 Harris, “Distributional Structure;” Coecke, “Text Structure.” 40 Coecke and Kissinger, Picturing Quantum. 41 Ibid.; Coecke, “Text Structure.” 42 Kartsaklis et al., “Lambeq;” Toumi, “Quantum Natural Language.” 38 Kartsaklis et al., “Lambeq.” 39 Harris, “Distributional Structure;” Coecke, “Text Structure.” 42 Kartsaklis et al., “Lambeq;” Toumi, “Quantum Natural Language.” 2.3 DisCoCat Until now, in the spirit of just-in-time learning, it has been left unsaid that a partially ordered monoid with the adjoints and expansion and contraction equations – again, a pregroup grammar – is also precisely a compact closed category, which includes an additional operation for parallel composition that allows the boxes to be lined up horizontally instead of vertically as in Figure 7.33 This operation is written as ⊗ f g and is read as “f while g” and provides the capability to account for multiple processes on disparate objects occurring simultaneously.34 For example, with a while operator, it becomes possible to represent multiple symbols simultaneously so that a potentially inﬁnite number of Figure 7s could be lined up horizontally. It is also true that boxes arranged in a horizontal line can represent words in sentences. Further, compact closed categories can leverage a special and elegant graphical representation known as string diagrams, which are closely related to the process diagrams of Figures 6 and 7.35 It turns out this is incredibly fortunate as string diagrams can be drawn to look remarkably like the graphs of mathematical linguistics. This makes the leap from linguistics to quantum very intuitive. Consider Figure 9 as the quantum form of Figure 8 that leverages a DisCoCat36 diagram, which is a speciﬁc type of string diagram commonly associated with QNLP research.37  31 Kartsaklis et al., “Lambeq.” 32 Coecke et al., “Foundations;” Coecke et al., “Qubits Speak.” 33 Coecke, “Text Structure.” 34 Coecke and Kissinger, Picturing Quantum. 35 Ibid. 36 DisCoCat is a portmanteau of three words: Categorical Compositional Distributional. It is a rather clever portmanteau since the order of reading composition is why the terms are reversed (think Cat ∘Co ∘Dis). 37 Coecke et al., “Foundations;” Coecke, “Text Structure.” First, this image comes from Karsaklis et al.38 Second, DisCoCat earned its name because these diagrams were developed by using category theory to unify distributional semantics – the linguistics and statistical research approach asserting that words used in the same contexts have similar meanings – and composition into one graphical tool.39 Notice how Figure 9 looks nearly identical to Figure 8 The primary diﬀerences are two fold: First the Figure 9: A DisCoCat diagram. 12  Ryder Dale Walton Figure 9: A DisCoCat diagram. 12  Ryder Dale Walton 12  Ryder Dale Walton 12 Figure 9: A DisCoCat diagram. 43 Augustine, Choice of the Will. 44 Feser, Five Proofs. 2.4.1 Theology Within the realm of theology, it is crucial to consider the epistemic priority of Christ, natural theology, and scholastic realism. It is, furthermore, helpful to consider the parallels between quantum philosophy and the mystic tradition. All these parts, perhaps seemingly disjointed to some, all play a role in observing “… God Said”: Toward a Quantum Theology of Creation  13 Figure 10: The principle of abstraction or the hierarchy of eternal truths. gy Figure 10: The principle of abstraction or the hierarchy of eternal truths. the spoken cosmos in a quantum circuit. When taken together, they are the ﬁrst ingredients to a quantum theistic interpretive framework. The investigation begins with an abbreviated journey to scholastic realism. Epistemology and natural theology will emerge on the way. the spoken cosmos in a quantum circuit. When taken together, they are the ﬁrst ingredients to a quantum theistic interpretive framework. The investigation begins with an abbreviated journey to scholastic realism. Epistemology and natural theology will emerge on the way. 2.4.1.1 The Augustinian Argument Saint Augustine, and those in his thought tradition, have argued for the existence of God on the grounds that abstract objects, also known as eternal truths, exist in the mind of a divine intellect – a notion commonly known as scholastic realism.43 While the present work does not aim to provide an argument for the existence of God, the Augustinian proof presupposes much of the metaphysical background needed to buttress this work’s primary contribution. In fact, the extension of this theological proof is the spine of this work. An abbreviated gloss of the argument, a distillation of Feser’s much more complete formulation, is as follows:44 1. Abstract objects like propositions, universal truths, mathematical numbers and concepts, and possib worlds all exist. Figure 10 provides one such example. 2. Realism, nominalism, and conceptualism are the three possible explanations for the existence of abstract objects. Realism holds that abstract objects have a real existence and are not reducible to anything material. Nominalism denies that abstract objects are real instead asserting that they are phantasms of language. Lastly, conceptualism asserts that abstract objects are real but are nothing more than constructs of the human mind. Of these three explanations, there are strong arguments in favor of realism. Additionally, there are insurmountable arguments against nominalism and conceptualism. Therefore, realism must be true. 3. There are, however, multiple ﬂavors of realism from which to choose such as Platonic realism, Aristotelian realism, and scholastic realism. Platonic realism is the belief that abstract objects exist in a third world – neither the world of the intellect nor the material world. This third world would be that of ideal forms. Aristotelian realism states that abstract objects exist both outside the mind in the essence of material objects as well as inside the mind performing abstract thought, making the existence of abstract object contingent on the existence of the material world and human minds. Scholastic realism holds that abstract objects exist inside the eternal, divine intellect. Of these three options, Scholastic realism has strong arguments in its favor. There are insurmountable arguments against Platonic realism and Aristotelian realism. Therefore, Scholastic realism is true. 4. There are strong arguments that the necessarily existing intellect in which abstract objects themselves exist is God’s own intellect. Therefore, God exists. 4. There are strong arguments that the necessarily existing intellect in which abstract objects themselves exist is God’s own intellect. Therefore, God exists. 2.4.1.2 Quantum Theology and the Mystic Tradition 2.4.1.2 Quantum Theology and the Mystic Tradition Quantum theory has made impacts on theology in the past. Though no previous major work has observed any implications of QNLP on theology, there have been meaningful insights that inform, at least indirectly, the present discussion. What follows is by no means an exhaustive treatment. Perhaps, the greatest exploration of quantum theology is that of O’Murchu, where he insists that the quantum era is marked by participation.45 It is essential to quantum theory that any observation of a quantum process substantively aﬀects the said process. While it is still hotly debated as to what the precise ramiﬁcations of this are, they are undoubtedly revolu- tionary.46 At the very least, nothing exists in isolation; therefore, it is impossible to observe in the sense of classical physics. It is, then, impossible by necessity for there to be any such thing as an observer or an observed. In the macroscopic world, it seems counter-intuitive that there are no observers or observed things, yet the realities of the quantum world, such as entanglement, not only validate O’Murchu’s claim but, in fact, necessitate it: “In the quantum worldview, nothing makes sense in isolation; basically, there are no bound- aries, and inﬂuences can emerge from several sources.”47 These liminal inﬂuences of which O’Murchu speaks cast doubt on the classical view of physics and, indeed other sciences as well, to the point that the validity of experiments conducted in tightly controlled environments comes into question. Alternatively, understanding must begin with totality rather than subdivision – isolated, distinct parts. In other words, understanding begins with the whole, which is greater – unknowable strictly by – the sum of its parts.48 Robert Barron notes that the participatory nature of reality is also found elsewhere in the theological tradition, so quantum theology is not strictly unique in this regard.49 Namely, participation is recognized by the greatest mystics throughout the millennia of recorded history. For example, John of the Cross truly understood the end of mysticism to be spousal union – the most intimate form of participation – with the Trinity.50 Indeed, insofar as creation reveals its trinitarian Creator, as asserted by natural theology and based on Paul’s rhetoric in Romans 1:18-20, creation’s parts must be inextricably linked and interconnected in relationship. 2.4.1.1 The Augustinian Argument is God’s own intellect. Therefore, God exists. 14  Ryder Dale Walton 14  Ryder Dale Walton The notion of two-ness, duality, or the pair has many diﬀerent physical representations. All of which are accidental. The substance of two has no physical form itself, but it is still very real from the perspective of scholastic realism as it exists inside the mind of God – it is not contingent on the existence of human minds to any degree. It is, therefore, an abstract object. Rather than delve into the intricacies of the Augustinian argument, it suﬃces to state that the proof is relevant here insofar as it asserts and supports the real existence of abstract objects, and that these abstract objects are physically instantiated into the material world from the divine intellect – the Logos and the basis of being itself. In other words, scholastic realism is a prerequisite for seeing the created world in quantum circuits. Now, there are two points to clarify here. First, grammar is an abstract object because grammar is mathematical. Second and therefore, the eeriness of mathematics – the interplay of abstract objects, to model, represent, and describe the workings of the material world to such a degree that they must be conceived of as real – also applies to grammar. This is signiﬁcant because it allows one to swap the word “grammar” for the word “math” in all theological arguments from mathematics without compromising said arguments. Grammar and mathematics are equally abstract, equally eerie, and equally real. Therefore, the grand insight of the Augustinian argument that abstraction tends heavenward and that abstract objects are real ﬁrmly undergirds this work’s primary contribution.  45 O’Murchu, Quantum Theology. 46 Aaronson, Quantum Computing. 47 O’Murchu, Quantum Theology, 33. 48 O’Murchu, Quantum Theology. 49 Barron, Christ. 50 John of the Cross, Spritual Canticle, stanzas 22-24; Barron, Christ. 51 Genesis 1:31. 2.4.1.2 Quantum Theology and the Mystic Tradition Further, that relationship must exist irrevocably since it is the eternal Godhead who creates and the Godhead who declares the creation good.51 For anything to be good, it must exhibit the invisible  45 O’Murchu, Quantum Theology. 46 Aaronson, Quantum Computing. 47 O’Murchu, Quantum Theology, 33. 48 O’Murchu, Quantum Theology. 49 Barron, Christ. 50 John of the Cross, Spritual Canticle, stanzas 22-24; Barron, Christ. 51 Genesis 1:31. 15 “… God Said”: Toward a Quantum Theology of Creation qualities of the one good God, who is the indivisible ground of being itself. Quantum mechanics, then, is a way of understanding physical reality’s participation in the metaphysical reality of the triune creator God’s inter- connected, relational, and eternal existence of love. The essential end of any spirituality is to change minds – metanoia.52 In the great Christian spiritual tradition, this is Christ’s proclamation that “The time is fulﬁlled, and the kingdom of God is at hand; repent and believe in the gospel.”53 In a quantum theology, this change of mind is an invitation to recognize the divine energy “impregnating” creation and to participate sacramentally and ecclesiastically in God’s relationship with all of creation.54 Furthermore, it is the call to right personal relationships at every cosmic level of being: people with people; people with animals; people with the earth; and ultimately, people with God. It ought to be emphasized that the quantum view of spirituality elevates relationships within the created order that are often understated. Indeed, O’Murchu’s view subverts the traditional view of humanity as the master of the created order. Instead, “Creation itself, and not we humans, is the primary narrator of the sacred story; we humans are invited to be the supreme listeners.”55 Insofar as quantum theology elevates the created order, it is not unlike primordial shamanism in its pursuit of the divine in the patterns and energy of nature. For better or worse, quantum theology’s tendency to observe scientiﬁc phenomena and subsume them into contemporary metaphysics is a necessary precursor to observing any theological ramiﬁcations of QNLP. Without the chan- ging of the mind to see that the underlying mathematics of language and physical processes reduce to quantum circuits – abstractions of quantum interactions – it would be diﬃcult to make the case that the created order is spoken into existence. Yet, quantum theology invites its hearers to precisely that metanoia – the elevation of the mind.  52 This word comes from the Greek word μετανοια, meaning beyond or over the mind. See Barron, Now I See. 53 Mark 1:15. 54 O’Murchu, Quantum Theology, 99. 55 Ibid., 211. 56 Coecke, “Theory of Everything,” 1. 57 Omnès, Quantum Philosophy. 58 Wang-Máscianica et al., “Distilling Text;” Coecke, “Theory of Everything;” Coecke and Paquette, “Categories.” 59 Omnès, Quantum Philosophy, 233. 57 Omnès, Quantum Philosophy. Q p y 58 Wang-Máscianica et al., “Distilling Text;” Coecke, “Theory of Everything;” Coecke and Paquette, “Categories.”  52 This word comes from the Greek word μετανοια, meaning beyond or over the mind. See Barron, Now I See. 53 Mark 1:15. 58 Wang-Máscianica et al., “Distilling Text;” Coecke, “Theory of Everything;” Coecke and Paquette, “Categories.” 59 Omnès, Quantum Philosophy, 233. 59 Omnès, Quantum Philosophy, 233. 2.4.2 Philosophy Apart from quantum theology, there also exists a branch of quantum philosophy, which seeks to reinvigorate classical philosophy given the discoveries of quantum mechanics. Quantum philosophical thought, as with its theological counterpart, also begins with “togetherness,” another word synonymous with participation, as Coecke aptly observes.56 Branching out from the root of togetherness, Omnès seeks to harmonize quantum mechanics with the philosophical discipline to inspire further inquiry into the nature of reality and its inter- play with the Logos.57 Even from an atheistic worldview, there is still a metaphysical urge to reevaluate the way humanity has come to understand philosophy, religion, and the scientiﬁc disciplines in light of quantum mechanics. This urge often manifests itself in a search for what is universal, be it in grammar, a theory of everything, or the unifying structures of category theory.58 It is also noteworthy that even in secular quantum philosophy, “realism is unassailable.”59 Abstract objects must exist in some real way because, even in the shadow of quantum mechanics, the observations, conclusions, and abstractions of common-sense perception will continue to hold true. It is these observations that constitute reality. Yet there is a wider and deeper reality available to the contemporary thinker who deigns to dive into the subatomic precisely because there is more to observe than meets the naked eye. There are still more abstractions to discover precisely because when there is more to see, there is more to infer and learn. Indeed, observation is the lynchpin of science, and science is, therefore, the discipline that studies reality. The Logos is deﬁned distinctly from reality in that it is the underlying rational principle through which all things operate; further, it is the epistemic reality through which all things ought to be interpreted. It is itself, 16  Ryder Dale Walton Figure 11: Reality and logos. Image is reconstructed based on Omnès, Quantum Philosophy, 268. Figure 11: Reality and logos. Image is reconstructed based on Omnès, Quantum Philosophy, 268. seemingly, abstract, and unobservable. It must, therefore, be within the realm of mathematics and logic. In quantum philosophy, the Logos exists in the abstract and permeates, or impregnates, reality. It is, thus, observable only insofar as its principles are embodied by a scientiﬁc object which is subject to some scientiﬁc discipline and study. Figure 11 provides a graphical depiction of this relationship. seemingly, abstract, and unobservable. 2.4.2.1 The Epistemic Priority of Christ The Christian mind holds that the Logos is Christ Himself.60 There is nothing created, interpretable, or even knowable apart from the mind of Christ.61 He, therefore, provides the epistemological basis for reality as described by Omnès in Figure 11. From the atheistic perspective, the Logos is ﬂeeting. Like a leaf blown on the wind, it is diﬃcult to grasp but, once seen, draws the gaze. The horizontal lines in Figure 8 demonstrate the parallelism between reality and the Logos, yet the lines are dashed because to the atheistic mind, the relation- ship is hazy and illy deﬁned. Still, it is important to acknowledge that the search for order is not simply a theistic drive and that realism is not simply a theistic apologist position. There are real parallels between reality and the Logos that beg further investigation. One such parallel is the reducibility of speech to quantum circuits as implemented by contemporary QNLP methods. 2.4.2 Philosophy It must, therefore, be within the realm of mathematics and logic. In quantum philosophy, the Logos exists in the abstract and permeates, or impregnates, reality. It is, thus, observable only insofar as its principles are embodied by a scientiﬁc object which is subject to some scientiﬁc discipline and study. Figure 11 provides a graphical depiction of this relationship.  60 John 1:1-5. 61 Colossians 1:15-18; Barron, Christ.  62 Feynman, “Simulating Physics;” Aaronson, Quantum Computing. 63 Whitﬁeld et al., “Electronic Structure Hamiltonians.” 64 Coecke and Kissenger, Picturing Quantum. 65 Qiskit, Textbook. 3 The Observation of the Participation With the preceding background laid as a foundation, it is time to participate in the scientiﬁc observation in three stages. First, a quantum circuit of a natural phenomenon is shown. Second, a couple of quantum circuits of grammar – linguistic phenomena – are also shown. The ﬁrst circuit depicts the opening words of Genesis, and the second depicts the opening words of the Gospel of John. Sentences from both Genesis and John’s Gospel are depicted because either one is ignored at peril. It is simply not ﬁtting to ignore the creation or the Logos. Third, and ﬁnally, the physical phenomenon is juxtaposed and compared with the simpler linguistic phenomenon, which is that from John. The journey through the next series of images on the foundation laid above terminates in the scientiﬁc observation of the metaphysical participation of the creation of the natural world through word. “… God Said”: Toward a Quantum Theology of Creation  1 17 Figure 12: Quantum circuit for electron interaction. This image is taken from Whitﬁeld, Biamonte, and Aspuru-Guzik’s “Electronic structure Hamiltonians” and is presented with no formatting alterations to preserve the integrity of their original work. Figure 12: Quantum circuit for electron interaction. This image is taken from Whitﬁeld, Biamonte, and Aspuru-Guzik’s “Electronic structure Hamiltonians” and is presented with no formatting alterations to preserve the integrity of their original work. 3.1 Stage One: Quantum Simulation of Natural Phenomena The most natural application of quantum circuits is, not surprisingly, in quantum simulation.62 It is, also unsurprisingly, the area where the largest amount of scholarly research is being conducted as it is of great interest to companies that deal in chemicals, materials, heavy industry, and stock market ﬁnance. From that body of research, a single example has been chosen for illustration. There are numerous other examples that could have been selected and are equally appropriate. Figure 12 depicts a quantum circuit that represents a real, physical interaction between two electrons. The exact nature of the interaction is unimportant for the present discussion, but is elaborated in Whitﬁeld, Biamonte, and Aspuru-Guzik.63 Before proceeding, a word should be oﬀered about the contents of Figure 12. Quantum circuits are based on simple logic circuits, which use the traditional logic gates – AND, OR, XOR, and so on. Most horizontal lines – wires – represent a qubit, though some are used to represent a register of classical binary bits. This classical register is either the bottommost or topmost wire depending on one’s preferred convention. The circuit is read from the left to the right with the state of the qubit being updated by passing through boxes as it goes. These boxes represent quantum gates, which are mathematical operations on unitary matrices. In short, this simply means that the boxes perform reversible – undoable – matrix operations on the qubits. They must be reversible because, in part, qubit states cannot be copied or cloned.64 This works perfectly well because matrices are nothing more than large collections of numbers used to represent the state of an object or system with the diﬀerent numbers representing diﬀerent values for attributes. The speciﬁc meaning of each gate in the circuits is well beyond the scope of the present discussion. Inquiring minds ought to reference the Qiskit Textbook for a structured approach to learning the speciﬁcs of quantum circuits and their gates.65 64 Coecke and Kissenger, Picturing Quantum. 3.2 Stage Two: Quantum Simulation of Grammar and Syntax Having now seen an example quantum circuit depicting a natural phenomenon and discussed its basic structure, the abstraction known as a quantum circuit is now something readily identiﬁable on sight. Next, then, DisCoCat diagrams for sentences from the opening portions of Genesis and John will be used to generate quantum circuits from grammar. These too will become a feast for the eyes. The ﬁrst DisCoCat diagram is introduced in Figure 13. It depicts the sentence “God said, ‘Let there be light’,” which is found in Genesis 1:3. Any discussion of creation through speech would certainly be remiss for not considering this grand utterance. Figure 13 takes the same basic form as that of Figure 9, and the explanation for DisCoCat diagrams provided in Figure 9 is equally applicable for Figure 13. For now, recall that the remaining wire that is unassociated between types written beneath two distinct boxes must be an s type 18  Ryder Dale Walton Figure 13: DisCoCat diagram for the sentence “God said, ‘Let There Be Light’”. This image is an original creation of the author using the python packages discopy and lambeq. For more information on these software tools, consult Toumi’s and Kartsaklis et al. (2022; 2021). Figure 13: DisCoCat diagram for the sentence “God said, ‘Let There Be Light’”. This image is an original creation of the author using the python packages discopy and lambeq. For more information on these software tools, consult Toumi’s and Kartsaklis et al. (2022; 2021). Figure 14: DisCoCat diagram for the sentence “The Word Became Flesh.” This image too is an original creation of the author using discopy and lambeq. Again, for more information on these software tools, consult Toumi’s, “Quantum Natural Language;” and Kartsaklis et al.’s, “Lambeq.” It is also noteworthy that this diagram can be simpliﬁed in preprocessing to drop the article adjective “The.” It is presented in this form so that the diagram maintains the exact structure of the biblical text. Figure 14: DisCoCat diagram for the sentence “The Word Became Flesh.” This image too is an original creation of the author using discopy and lambeq. 3.2 Stage Two: Quantum Simulation of Grammar and Syntax In this representation, the classical bit register is the wire drawn across the bottom. Note also that the diagram is long enough that it is drawn across two rows. Figure 16: “The Word” as a quantum circuit. This image is original to the author. Because this sentence is radically simpler, it is better for juxtaposition in with Figure 12 in Figure 17. Figure 16: “The Word” as a quantum circuit. This image is original to the author. Because this sentence is radically simpler, it is better for juxtaposition in with Figure 12 in Figure 17. Having introduced Figures 13 and 14, it is time to convert them into quantum circuits and examine them anew. Figures 15 and 16 are the quantum circuit forms of the DisCoCat diagrams found in Figures 13 and 14, respectively. Both Figures 15 and 16 are introduced and discussed as a pair. Having introduced Figures 13 and 14, it is time to convert them into quantum circuits and examine them anew. Figures 15 and 16 are the quantum circuit forms of the DisCoCat diagrams found in Figures 13 and 14, respectively. Both Figures 15 and 16 are introduced and discussed as a pair. Here, it is crucial to observe that while quantum circuits may have varying degrees of complexity – some require more qubits, so they grow taller vertically, and others require a greater gate depth so that they grow wider horizontally – some subset of the same mathematical building blocks are used in all quantum circuits regardless of complexity. Speciﬁcally, Figures 15 and 16 contain the familiar rotational gate, R, and the Hadamard gate, H, that ﬁrst appeared in Figure 12.67 3.2 Stage Two: Quantum Simulation of Grammar and Syntax Again, for more information on these software tools, consult Toumi’s, “Quantum Natural Language;” and Kartsaklis et al.’s, “Lambeq.” It is also noteworthy that this diagram can be simpliﬁed in preprocessing to drop the article adjective “The.” It is presented in this form so that the diagram maintains the exact structure of the biblical text. for the sentence to be grammatical. Figure 13, therefore, represents a grammatical utterance that is ﬁt for generating a quantum circuit. Figure 14 is included as an additional example for two reasons. First, it is from the opening lines of John’s Gospel, “The Word became ﬂesh,” which is the section that essentially functions as the creation account – the Genesis – of the New Testament.66 These two accounts are, then, meant to be contemplated together with the John account explicitly describing the creation through the lens of the real embodiment of the Logos, which is pertinent relative to the prior discussion of quantum philosophy. Second, this sentence produces a simpler quantum circuit, which is more instructive in stage three of the observation. 66 (1:14) “… God Said”: Toward a Quantum Theology of Creation 19 Figure 15: “Light” as a quantum circuit. This image is original to the author. The format presented here is that of Qiskit, which admittedly looks diﬀerent from Figure 12; however, the diﬀerences are strictly limited to formatting conventions. In this representation, the classical bit register is the wire drawn across the bottom. Note also that the diagram is long enough that it is drawn across two rows. Figure 16: “The Word” as a quantum circuit. This image is original to the author. Because this sentence is radically simpler, it is better for juxtaposition in with Figure 12 in Figure 17. Figure 15: “Light” as a quantum circuit. This image is original to the author. The format presented here is that of Qiskit, which admittedly looks diﬀerent from Figure 12; however, the diﬀerences are strictly limited to formatting conventions. In this representation, the classical bit register is the wire drawn across the bottom. Note also that the diagram is long enough that it is drawn across two rows. Figure 15: “Light” as a quantum circuit. This image is original to the author. The format presented here is that of Qiskit, which admittedly looks diﬀerent from Figure 12; however, the diﬀerences are strictly limited to formatting conventions. 67 Qiskit, Textbook. 3.3 Stage Three: The Juxtaposition What remains now is to juxtapose Figure 12 with Figure 16 to make a quick comparison of their content. Figure 17 shows the result. 67 Qiskit, Textbook. Figure 17: The juxtaposition of physical and grammatical phenomena. 20  Ryder Dale Walton 20  Ryder Dale Walton 20 Figure 17: The juxtaposition of physical and grammatical phenomena. Ignoring the formatting diﬀerences, which is strictly a diﬀerence in the technology used in Whiteﬁeld, Biamonte, and Aspuru-Guzik and the present work, the graphical, material resemblance is uncanny.68 Now, the question remains: What is it about the juxtaposition of these two images that leads the metaphysically inclined mind to conjecture that the physical, ordered world is created by word? Certainly, both images are made up of lines and squares, and many of the squares have the same letters in them, but it is more than that. It is the line and the square as a depiction of abstract mathematical objects that inspire the connection of grammar to creation. Both grammar and creation share an abstraction – a metaphysical super-structure – like that of duality in Figure 10. Namely, that abstraction is quantum-ness or quantum-native-ness itself represented by the unifying abstractor of the quantum circuit. That speech – the Word – is both Logos and quantum should be no surprise given that which was spoken – reality and the cosmos itself – is quantum. Because of this quantum-native-ness, discoverable only through the scientiﬁc advancements of QNLP, creation through speech is observable – meaning visible – by the participation of the naked eye in graphical format for the ﬁrst time with no divine revelation required.  68 Whitﬁeld et al., “Electronic Structure Hamiltonians.” 4 Conclusion These diagrams, then, can be processed via software to create quantum circuits. With that development, it is now plainly visible that the natural world and grammar both share the abstraction of the quantum circuit. Next theology and philosophy enter the picture. First, assuming natural theology – the idea that the natural world reveals the characteristics of the Creator – is a given. If the qualities of the Creator cannot be seen in the creation, any enterprise searching for truth through scientiﬁc means, or simply the senses in general is doomed to failure. Next, if the natural world reveals the qualities of the Creator, then abstraction is the process by which the qualities of the Creator may be ascertained. By examining the Augustinian proof for the existence of God, it becomes evident that a form of scholastic realism provides the best explanation for the existence of abstract objects – the outputs of the abstraction process – and their independence from created minds. There must be a supreme mind that holds these abstract objects in existence apart from the existence and experience of human consciousness. Whether that is the God of Christianity, another religion, or con- sciousness itself is immaterial for this portion of the argument. All that matters is that abstract objects have some kind of real existence. A speciﬁc quantum theology begins to emerge at this point. Theoretical quantum physics demonstrates the interconnectedness of the world so that everything is a participant in the ongoing unfolding of reality. The implication here is that classical experimentation is quite limited in its explanatory power, and is, certainly, metaphysically useless when it comes to discerning the nature of the world since it neither accurately reﬂects the nature of the world nor its structure. Furthermore, classical experimentation creates the great sin of separation. Creation exists as a great orchestra of resounding unity, and any attempt to dissect it into its individual parts creates a skewed view of the world. Unity is where the world is seen as functioning in its true state, and in this unity is where understanding becomes possible. The forest can no longer be lost for the trees. The great mystics of all traditions were aware of this shamanic truth. 4 Conclusion From the outset, this work has endeavored to show primarily that QNLP has a unique theological and philosophical ramiﬁcation in that it allows the concept of creation through speech in accordance with the Logos, divinely revealed in Genesis 1 and John 1, to be inferred – even observed and participated with – visibly from the discipline of quantum computing alone. No divine revelation is required. This is the principal contribution of this work. There is, however, a great deal of background and prior research required to substantiate this contribution. It starts, in the strictly scientiﬁc sense, with the fact that the world is not classical. It is instead quantum mechanical, which brings with it a certain set of metaphysical claims as to the nature of reality and the fact that the very act of observation itself is a participation in the experiment. There are no longer subjects or objects, only participants. Further, since the world is quantum mechanical, the underlying mathematics of the universe are also quantum. This mathematics can be described in two camps: quantum mechanics itself – the nuts and bolts of matrix mathematics, Kronecker products, and other approaches – and category theory. The former leads to the development of quantum simulation and circuits and the latter to an abstrac- tion ﬁt for describing many disciplines and realities within a singular, higher-order mathematical abstraction. Category theory is itself strong enough to model the theistic universe insofar as it provides a framework for “… God Said”: Toward a Quantum Theology of Creation 21 mathematically representing symbols and rituals to instantiate them from the materials of heaven-stuﬀand earth-stuﬀ. This is particularly appropriate for sacramental theologies but can represent any symbolic world- view so long as the structure of the cosmos in that worldview is suﬃciently understood. An introduction to this potential avenue of research and an invitation to explore it is the secondary contribution of this work. Given the quantum nature of creation, it remained to establish the quantum nature of language. This begins by establishing a mathematical system to model grammar, which ﬁrmly roots language in the reality of mathematics using types that represent something akin to matrices. The simplest choice for this is that of pregroups. Once this has been established it is a small step to add boxes, wires, and a few postulates to add structure and form DisCoCat diagrams. 69 Entrustedtothedirt, “Wonder of Language;” Job 38:4. 22  Ryder Dale Walton 22  Ryder Dale Walton 4 Conclusion Christian mystics are chief among these as contemplation of the eternal relationship of the Trinity has led them to the understanding that all of creation exists in relationship with itself, with the Creator, and with humanity. To be, whether abstract objects or material objects, is to be in a relationship. Because it is now wrestling with the quantum realm, science is now beginning to catch up to the Christian mystical tradition. Finally, the inquisitive mind returns to the quantum circuits and takes a moment to contemplate a couple of examples: one representing a physical process, and another representing grammatical, human language. It is at this point that the Christian account of the Creator begins to be the most ﬁtting, for the Christian God is one that exists in eternal relationship – perhaps eternal entanglement – and creates through the spoken word, that is the Word – the Logos. Any secular philosopher and any theologian alike seeks to understand the structure of reality and the underlying principle – the Logos – that makes it understandable. It is the very fact that the material order and the spoken order are really the same order that one observes – really participates in – in the quantum circuits with no divine revelation required. This is that principle. It is in this counterintuitive space, opened by viewing the outputs of a scientiﬁc experiment with a metaphysical mind, that the creation of the material and the creation of the grammatical as ascended into the same abstraction become interchangeable: “Were you there when I laid the foundation of grammar?”.69 It is in this space that the continued speech of God creates and holds all lovingly in being even now, where the dancing of our electrons and the beats of our hearts answer Him. References Entrustedtothedirt. “The Image of God and the Wonder of Language.” Entrusted to the Dirt. (2020, August 21). https://entrustedtothedirt. de Felice, Giovanni. “Categorical Tools for Natural Language Processing.” arXiv. (2022, December 13). doi: 10.48550/arXiv.2212.06636. Entrustedtothedirt. “The Image of God and the Wonder of Language.” Entrusted to the Dirt. (2020, August 21). https://entrustedtothedirt. Entrustedtothedirt. “The Image of God and the Wonder of Language.” Entrusted to the Dirt. 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https://openalex.org/W2460718529	https://krex.k-state.edu/bitstream/2097/35180/1/Commerford_et_al-2016-Ecology_and_Evolution.pdf	English	null	High dissimilarity within a multiyear annual record of pollen assemblages from a North American tallgrass prairie	Ecology and evolution	2,016	cc-by	10,945	ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 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. Ecology and Evolution 2016; 6(15): 5273– 5289 doi: 10.1002/ece3.2259 doi: 10.1002/ece3.2259 timing of these changes and the factors inﬂuencing those changes at various spatial scales are still unknown. There- fore, both the composition and biodiversity of grasslands over time and the role that biotic and abiotic factors have played in structuring them remain unanswered questions. A long-term perspective is essential to capture the slow processes thought to be important in North American grasslands, such as megadroughts, grazing from large her- bivores, and changes in ﬁre regimes. Keywords Keywords Fire, grassland, Great Plains, herbivory, pollen, Tauber traps. Keywords Fire, grassland, Great Plains, herbivory, pollen, Tauber traps. Grassland vegetation varies in composition across North America and has been historically inﬂuenced by multiple biotic and abiotic drivers, including ﬁre, her- bivory, and topography. Yet, the amount of temporal and spatial variability exhibited among grassland pollen assemblages, and the inﬂuence of these biotic and abiotic drivers on pollen assemblage composition and diversity has been relatively understudied. Here, we examine 4 years of modern pollen assemblages collected from a series of 28 traps at the Konza Prairie Long-Term Ecological Research Area in the Flint Hills of Kansas, with the aim of evaluating the inﬂu- ence of these drivers, as well as quantifying the amount of spatial and temporal variability in the pollen signatures of the tallgrass prairie biome. We include all terrestrial pollen taxa in our analyses while calculating four summative metrics of pollen diversity and composition – beta-diversity, Shannon index, nonarbo- real pollen percentage, and Ambrosia:Artemisia – and ﬁnd different roles of ﬁre, herbivory, and topography variables in relation to these pollen metrics. In addi- tion, we ﬁnd signiﬁcant annual differences in the means of three of these met- rics, particularly the year 2013 which experienced high precipitation relative to the other 3 years of data. To quantify spatial and temporal dissimilarity among the samples over the 4-year study, we calculate pairwise squared-chord dis- tances (SCD). The SCD values indicate higher compositional dissimilarity across the traps (0.38 mean) among all years than within a single trap from year to year (0.31 mean), suggesting that grassland vegetation can have different pollen signatures across ﬁnely sampled space and time, and emphasizing the need for additional long-term annual monitoring of grassland pollen. Correspondence p Julie L. Commerford, Department of Geography, Seaton Hall, Kansas State University, Manhattan, KS 66506. Tel: +1 507 766-9231; E-mail: jcomm@ksu.edu Funding Information Funding Information Division of Behavioral and Cognitive Sciences (Grant/Award Number: “0955225”, “1558228”) Division of Graduate Education (Grant/Award Number: “0841414”) Division of Environmental Biology (Grant/Award Number: “0823341”). g Division of Behavioral and Cognitive Sciences (Grant/Award Number: “0955225”, “1558228”) Division of Graduate Education (Grant/Award Number: “0841414”) Division of Environmental Biology (Grant/Award Number: “0823341”). Received: 1 February 2016; Revised: 23 May 2016; Accepted: 27 May 2016 Ecology and Evolution 2016; 6(15): 5273– 5289 Introduction For example, much work has been made to calibrate pollen with vegetation by calculating pollen productivity of select plant taxa in Europe (Soepboer et al. 2007; Brostrom et al. 2008; Mazier et al. 2008) and in the United States (Sugita et al. 2006; Commerford et al. 2013). In addition, calibrations of pollen with climate have been conducted in a variety of ecosystems, including grasslands (Gajewski et al. 2002; Minckley et al. 2008; Tonello and Prieto 2008). reason for this problem is that pollen is not produced in proportion to plant abundance on the landscape (Sugita 1994). One way to overcome this limitation is by calibrat- ing modern pollen assemblages with site features such as vegetation, climate, ﬁre, herbivory, or topography. For example, much work has been made to calibrate pollen with vegetation by calculating pollen productivity of select plant taxa in Europe (Soepboer et al. 2007; Brostrom et al. 2008; Mazier et al. 2008) and in the United States (Sugita et al. 2006; Commerford et al. 2013). In addition, calibrations of pollen with climate have been conducted in a variety of ecosystems, including grasslands (Gajewski et al. 2002; Minckley et al. 2008; Tonello and Prieto 2008). Modern pollen samples are acquired in several ways, including surface sediments from lakes or ponds, moss polsters, or Tauber traps (Tauber 1974). Tauber traps have been successfully deployed over a variety of forested landscapes in Poland, Norway, Finland, Latvia, Czech Republic, Switzerland, Greece, and Bulgaria in multiyear studies of pollen production through the European Pollen Monitoring Programme (Giesecke et al. 2010). The goal of these studies has been to calibrate pollen deposition with vegetation cover and examine annual and seasonal variability (Hicks 1985, 2001; R€as€anen et al. 2004; Gie- secke and Fontana 2008; Bjune 2014). Tauber traps are typically made of thick plastic, are cylindrical in shape, and are approximately 25 cm tall by 15 cm in diameter (Fig. 1). An opening of 5 cm in diameter at the top of the trap allows pollen to enter and remain trapped for the duration of the sampling period. Here, we present a 4-year annual record of modern pollen assemblages from a tallgrass prairie in Kansas, with the aim of quantifying temporal and spatial variation in the context of biotic and abiotic drivers. Introduction We evaluate annual grassland pollen assemblages with several different ﬁre, herbivory, and topography variables to achieve three main objectives: (i) assess the inﬂuence of these variables on the pollen assemblages; (ii) quantify the degree of interannual variability among the pollen assemblages; and (iii) determine whether the pollen assemblages exhibit higher spatial variation (between sample locations) or temporal variation (within sample locations). To do this, we calculate four summative metrics from each pollen assemblage to represent their composition and/or diver- sity, and then quantify the degree of explanation of each of these metrics by the environmental variables. We then statistically compare the differences in these metrics across the 4 years of sampling. Finally, we evaluate the degree of dissimilarity between pollen assemblages across the study area and through time by calculating squared- chord distance between each pollen assemblage and all other assemblages. By doing so, we provide greater insight into the factors that drive grassland pollen assemblage composition and diversity, as well as highlight the amount of variation that exists among grassland pollen assemblages across space and through time. p g p Two recent examples from Europe demonstrate the sig- niﬁcant value of these multiyear pollen records. In a birch-pine forest in Norway, nine Tauber traps were used to collect pollen annually from 2004 to 2012 (Bjune 2014). After 8 years of sampling, there was higher inter- annual variability in pollen productivity than after only 2 years of sampling. These results suggested that multi- year, annual records of pollen deposition are necessary to understand the degree of variability in pollen representing a landscape over time. In a similar study in a birch forest in Finland, 6 years of pollen at four different Tauber traps were collected and compared with corresponding years of meteorological data (Autio and Hicks 2004). Here, the deposition of Pinus (pine) pollen was positively correlated with July temperature. These ﬁndings demonstrated that climate conditions, particularly temperature, could be used to predict pollen production for the following year. These studies of annual pollen production reveal the need for caution in overinterpreting environmental conditions at the onset of changes in paleoenvironmental records as increases in individual pollen taxa could be responses to rapid shifts in climate (Minckley et al. 2012a). Introduction Grasslands occupy 24% of Earth’s land area and con- tribute to global food production through row-crop agri- culture and grazing or pasture lands. Because grasslands experience high spatial and temporal variability in cli- mate, they are predicted to be especially vulnerable to future climate change (IPCC 2014). Grasslands in North America are particularly at risk because they are currently spatially restricted and regularly experience severe droughts such as the Dust Bowl of the 1930s (Cook et al. 2010). Other similar long-term drought events have occurred in this region throughout the Holocene (the past 11,500 years), changing the species composition and extent of this biome (Clark et al. 2001; Umbanhowar et al. 2006; Grimm et al. 2011). However, the precise Pollen from lacustrine sediment cores records how veg- etation responded to those processes on relevant time- scales of several decades to millennia (Clark et al. 2001; Brown et al. 2005). While pollen from sediment cores can provide a unique source of information about past vege- tation on a landscape, it has been difﬁcult to obtain quantitative estimates of vegetation cover. The main 5273 High Dissimilarity With in Grassland Pollen J. L. Commerford et al. While multiyear studies have been important for improving vegetation and climatic interpretations from pollen, to our knowledge there have been few similar studies in North America. One North American study examined annual modern grassland pollen records, but focused on spatial differences among grassland types and only collected pollen for one or 2 years (Hoyt 2000). Most importantly, little work has been made to calibrate factors aside from climate or vegetation, such as ﬁre, her- bivory, or topography. These factors are of major impor- tance in grasslands (Knapp et al. 1999; Craine and McLauchlan 2004; Grimm et al. 2011). Consequently, there is a clear need to understand the quantitative rela- tionships among ﬁre, herbivory, and topography and pre- sent-day grassland composition. A detailed examination of how those factors inﬂuence pollen deposition on an annual basis is required to understand these relationships in the distant past. reason for this problem is that pollen is not produced in proportion to plant abundance on the landscape (Sugita 1994). One way to overcome this limitation is by calibrat- ing modern pollen assemblages with site features such as vegetation, climate, ﬁre, herbivory, or topography. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Study area Konza Prairie Long-Term Ecological Research Area (here- after “Konza”) is a 3487-hectare preserve of native tall- grass prairie in the Flint Hills of northeastern Kansas (Fig. 1). The Flint Hills contains the largest contiguous 5274 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. 5 9 7 6 3 2 4 8 1 10 24 23 22 11 21 25 18 20 16 15 14 17 13 19 12 26 27 Konza Pollen traps Konza experimental watersheds Flint Hills 0 1 2 Kilometers Photo: Bing Imagery, 2010 Flint Hills Figure 1. Trap locations within the Konza Prairie Long-Term Ecological Research Area. Konza is located within the Flint Hills ecoregion (inset map). A typical trap is pictured in the photograph. Konza Flint Hills Konza 0 6 19 15 Figure 1. Trap locations within the Konza Prairie Long-Term Ecological Research Area. Konza is located within the Flint Hills ecoregion (inset map). A typical trap is pictured in the photograph. 5275 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. environmental variables at each sample location. Most environmental variables for each sample location were obtained from Leys et al. (2015), which examined macro- scopic charcoal from this same set of traps for the same years (2011–2014). The ﬁre metrics we included were mean ﬁre return interval, ﬁre return interval, ﬁre fre- quency, time since last ﬁre, regional area burned within the Flint Hills ecoregion, local area burned within Konza, and area burned within 500 m of the trap. Topography metrics we included from Leys et al. (2015) were eleva- tion, presence of bare soil around the trap, presence of bison manure (as a qualitative proxy for presence/absence of bison), and presence of limestone rocks (within 500 m of the trap). To supplement the above variables, we used bison density data calculated as amount of time spent by bison within 500 m of each trap via GPS collars and pre- viously reported in Gill et al. (2013). Data analysis Twenty-eight modiﬁed Tauber traps (Tauber 1974) were placed on Konza in October 2008. Traps were placed stratiﬁed-randomly to cover both grazed and ungrazed areas, different burning treatments, and upland and low- land areas. Traps were placed at least 500 m apart. The trap contents were collected in October 2009, 2010, 2011, 2012, 2013, and 2014 (at the end of the growing season). Each trap was rinsed in the ﬁeld with deionized water and poured into an empty jug. In the laboratory, the con- tents were ﬁltered through cellulose paper and soaked with glacial acetic acid to prevent the growth of mold or fungi. Each ﬁlter paper was processed and prepared for pollen analysis using standard techniques (Faegri and Iversen 1989) with a modiﬁcation made for dissolving the ﬁlter paper based on the European Pollen Monitoring Programme protocol. Each pollen sample was mounted in silicone oil, examined under a light microscope, and counted to a minimum sum of 300 terrestrial grains. All pollen grains were identiﬁed to the ﬁnest possible taxo- nomic resolution, generally following McAndrews et al. (1973). This article focuses speciﬁcally on the pollen data collected in 2011, 2012, 2013, and 2014. The pollen data from 2009 and 2010 are published in Gill et al. (2013). Raw pollen counts were converted to percentages using a pollen sum that included all terrestrial taxa and analyzed in Tilia 2.0 (Grimm 1993). High variability in volume of water used to wash the traps and ﬁlter their contents pre- cluded calculations of absolute pollen inﬂux rate, although the protocol could be amended in future years to allow that calculation. One trap was excluded from the analyses because it contained no pollen in 2011 and 2012. To initially examine the degree to which the environmen- tal variables inﬂuenced the multivariate pollen samples, we conducted a canonical correspondence analysis (CCA) (Ter Braak 1986) using Canoco 5 Software (Ter Braak and Smilauer 2012). The CCA suggested some inﬂuence of certain environmental variables on the multivariate pollen assemblages, but overall low explanation (highest eigenvalue = 0.06). Thus, we determined that a collective value of diversity or composition for each sample based on all of the pollen taxa would be more useful for directly assessing the inﬂuence of the environmental variables. We chose to summarize each pollen sample (each trap from each year) using four different metrics included in a regression analysis and compared across all 4 years. Study area We also calculated total precipitation and mean average, maximum, and minimum temperature for the months May through August for each year, based on data from the Konza Prairie weather station (KPBS 2015), to examine any covariance of these factors with the 4 years of pollen data. All three temperature variables were found to exhibit no signiﬁcant annual differences for the 4 years covered in this study and so were not pursued further. Precipitation was included because of signiﬁcant annual differences. unplowed tract of prairie remaining in North America (Knapp et al. 1999), and the parent material consists of alternating layers of limestone and mudstone. Konza is divided into watershed-level experimental units that were delineated in 1981, and each unit has a speciﬁed grazing and burning treatment. Each watershed is burned every 1, 2, 3, 4, or 20 years. A herd of approximately 300 native bison have resided on Konza since its introduction in 1987, and are restricted to the area outlined in black on Figure 1. The preserve is ﬂoristically diverse, with 597 different plant species: 86 species of grasses, 409 species of forbs, and 59 woody species (Towne 2002). Konza was chosen for this study for a number of reasons: (i) It has never been plowed and so is the best present-day proxy for grasslands prior to the onset of modern agriculture; (ii) Its ﬂoristic diversity provides an ideal location for captur- ing differences in pollen representation among grassland plants; (iii) The different burning and grazing treatments facilitate direct comparison between pollen assemblages in watersheds that are burned at different frequencies, in addition to grazed versus ungrazed areas; and (iv) Its pur- pose as a long-term ecological research area enables a multiyear study where pollen traps can remain in-place for several years with minimal disruption. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Equation 1: Shannon index Shannon index has been found to be a robust measure of vegetation diversity when used on pollen assemblages (Matthias et al. 2015). Beta-diversity was calculated via a detrended canonical correspondence analysis (DCCA), constrained by year. This metric computes the ratio between the sample diversity at a given trap for 1 year compared to the sample diversity at that same trap for the other 3 years. Nonarboreal pollen percentage was cal- culated by totaling the abundance proportions of all grass and forb pollen in each sample. Ambrosia:Artemisia was calculated as the ratio of Ambrosia:Artemisia pollen per- centages in each sample and has been suggested to be an indicator of grassland type (tallgrass vs. shortgrass) (Mor- ris 2013). To evaluate the dissimilarity between each pollen sam- ple, we calculated pairwise squared-chord distances. The squared-chord distance (SCD) metric (Overpeck et al. 1985) incorporates all pollen taxa in each sample and essentially computes the component distance between sample pairs, such that pairs with higher SCD values are more dissimilar, while pairs with lower SCD values are more similar. SCD has long been used in pollen studies to detect a matching modern pollen sample for a fossil pollen sample (Williams et al. 2009; Minckley et al. 2012b; McLauchlan et al. 2013). It has also been used to compare dissimilarity among pollen samples across a modern study area (Hoyt 2000; Gavin et al. 2005; Minck- ley et al. 2008), such as in this study. We calculated SCD in R Statistical Software (R Core Team 2015), using the Analogue package (Simpson 2015). Please see Appendix S1 for speciﬁc code. To assess which environmental variables inﬂuenced each of the four summative pollen metrics, individual multiple regressions were performed. All summative met- rics and environmental variables were rescaled between 0 and 1 using the “scales” package in R (Wickham 2016). Each regression used one of the four summative metrics (Shannon index, beta-diversity, nonarboreal percentage, and Ambrosia:Artemisia) as the response variable, and all of the previously mentioned environmental variables as the explanatory ﬁxed-effect variables. Random effects for trap and year were also included in each regression model and tested for signiﬁcance. For each response variable, this was performed by creating a full model of all ﬁxed effects (environmental variables) and random effects (for trap and year). Equation 1: Shannon index Then, the random effects of trap and year were each removed individually from the full model, and the resulting model was statistically compared to the full model. The regression models were built using lme4 pack- age (Bates et al. 2015) in R. Comparisons between the full models and the models with random effects removed were conducted using the stats package in R (R Core Team 2015). Please see Appendix S1 for speciﬁc code. Data analysis The four different metrics calculated were as follows: (i) Shan- non index, (ii) beta-diversity, (iii) nonarboreal pollen To evaluate the effects of ﬁre, grazing, and topography on the pollen assemblages, we measured several different 5276 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. each of the other 3 years (at P < 0.05). Finally, an analy- sis of covariance (ANCOVA) was conducted to test the covariance of total growing season precipitation and year for each of the four summative metrics. The temperature variables mentioned earlier (minimum, average, and max- imum temperature) were excluded from the analysis because they were not signiﬁcantly different across the 4 years of data. All of these analyses were conducted in R statistical software (R Core Team 2015), using the stats package for the regression and ANCOVA (R Core Team 2015), and the PGIRMESS package (Giraudoux 2014) for the ANOVAs and post hoc comparisons. Please see Appendix S1 for speciﬁc code. percentage, and (iv) ratio of Ambrosia:Artemisia pollen. Shannon index was calculated using the standard equa- tion for Shannon diversity (Equation 1), where p is the proportion of the sample belonging to pollen taxa i. H0 ¼ X R i¼1 pi ln pi ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Pollen assemblages among traps and years For each trap, each bar signiﬁes one collection year, beginning with 2011 at the top and 2014 at the bottom. most abundant taxa for all 4 years. In addition, some of the less abundant (but still common) taxa also exhibit trends. Xanthium pollen is higher at Trap 10 than at any of the other traps for 2011, 2012, and 2013. Artemisia is highest in 2011, 2012, and 2013 at Trap 22 compared to all the other traps but is higher at Trap 17 in 2014. Thus, trap locations contribute to the types of pollen captured. the trap. The random effects of trap location and year also vary in signiﬁcance for the four summative metrics. Trap location signiﬁcantly inﬂuences Shannon index, NAP%, and Ambrosia:Artemisia. Year signiﬁcantly inﬂu- ences Shannon index only. Beta-diversity is not signiﬁ- cantly inﬂuenced by trap location or year. y y y There are signiﬁcant differences among the 4 years for three of the four summative pollen metrics (Shannon Index, beta-diversity, and NAP%) demonstrated by the Kruskal–Wallis tests (Fig. 3). However, the amount of variation depends at least to some degree on the metric being used. 2013 stands out as either the highest or lowest year for each of the four metrics. Shannon Index is lowest in 2013 (median 1.82). Beta-diversity is also lowest in 2013 (median 0.84). Both the NAP% and Ambrosia:Arte- misia are highest in 2013 (median 73.95 and 23.25, respectively). 2013 is signiﬁcantly different (at P < 0.05) from 2011 in all four metrics. However, it is not signiﬁ- cantly different from 2012 in NAP% and beta-diversity or from 2014 in Shannon Index. 2011 and 2014 were not signiﬁcantly different from each other in any of the four metrics. In addition, Ambrosia:Artemisia did not show any signiﬁcant differences among the 4 years of data. Total growing season precipitation signiﬁcantly co-varies Pollen assemblages among traps and years Percent abundance of pollen taxa varies signiﬁcantly among the 27 Tauber traps at the Konza study area from 2011, 2012, 2013, and 2014 (Fig. 2). The number of dif- ferent pollen taxa from each trap/year ranges from 11 to 32, averaging 22 taxa. The six most abundant pollen taxa found in the traps on average are (in order from the highest): Ambrosia (31.1%), Quercus (18.9%), Poaceae (9.6%), Undifferentiated Asteraceae (8.9%), Cupressaceae (5.7%), and Amaranthaceae (4.1%). Four of these taxa are nonarboreal (Ambrosia, Poaceae, undifferentiated Asteraceae, and Amaranthaceae), and two of these taxa are arboreal (Quercus and Cupressaceae). We also compared differences in the summative metrics across all 4 years of data. To do this, a Kruskal–Wallis ANOVA test was conducted on each of the four metrics across the 4 years, to determine whether the means for each year were signiﬁcantly different overall (at P < 0.05). A Kruskal–Wallis post hoc pairwise comparison tested for signiﬁcant differences between the mean of each year and The identity of the most abundant pollen taxa varies locally from trap to trap, although certain traps exhibit more variation across the 4 years of data than other traps. At Trap 2 and Trap 8, Quercus is consistently the most abundant taxa for all 4 years of data. Conversely, at Trap 1, 11, 12, 13, 21, and 25, Ambrosia is consistently the 5277 High Dissimilarity With in Grassland Pollen J. L. Commerford et al. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Trap 20 40 60 80 Asteraceae undiff 20 40 60 80 100 Ambrosia 20 Artemisia Iva 20 Xanthium 20 Amaranthaceae 10 Cyperaceae 20 40 Poaceae 10 Carya 10 Cornus 20 Cupressaceae 10 Pinus 20 40 60 Quercus 10 Rhus Percent abundance Figure 2. Pollen abundance (%) for the 6 most abundant taxa and other key taxa. Nonarboreal taxa are shown in brown, and arboreal taxa are shown in green. For each trap, each bar signiﬁes one collection year, beginning with 2011 at the top and 2014 at the bottom. Figure 2. Pollen abundance (%) for the 6 most abundant taxa and other key taxa. Nonarboreal taxa are shown in brown, and arboreal taxa are shown in green. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Inﬂuence of ﬁre, grazing, and topography variables The environmental variables known to inﬂuence grass- land vegetation – ﬁre frequency, bison density, and ele- vation – are somewhat reﬂected in pollen assemblages produced by grassland vegetation. Each of the four summative pollen metrics is inﬂuenced by different environmental variables, which is logical given that some of the calculated pollen metrics assess diversity while others focus on pollen composition (Table 1). Previous work in other regions has suggested that there is no single best metric for assessing pollen diversity and composition (Weng et al. 2007; Birks et al. 2016), and our results indicate that the same is true for grass- lands in North America. However, some variables (e.g., area burned within Konza and area burned within the Flint Hills) signiﬁcantly contribute to more than one pollen metric. The role of environmental variables on pollen metrics Summative pollen metrics Beta-diversity Shannon NAP% Ambrosia:Artemisia Fixed effects Elevation – – – – BareSoil – – – – LimestoneRocks – – – – BisonManure – – – – Bison Density – 0.25* – – Area Burned 500 m – – – –0.14* Area Burned Flint Hills 0.45* – 0.13 – Fire Return Interval – – – – Mean Fire Return Inter. – – – – FireFrequency – – – – TimeSinceLastFire – – – – Area Burned Konza 0.29* – 0.16* – Random effects Trap Not signiﬁcant Signiﬁcant (P<0.001) Signiﬁcant (P<0.001) Signiﬁcant (P<0.001) Year Not signiﬁcant Signiﬁcant (P<0.05) Not signiﬁcant Not signiﬁcant Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.05; Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.01; *Indicates the ﬁxed- effect variable is signiﬁcant at P < 0.001. Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.05; Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.01; Indicates the ﬁxed- effect variable is signiﬁcant at P < 0.001. ct variable is signiﬁcant at P < 0.05; Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.01; *Indicates the ﬁxed ant at P < 0.001. with year for beta-diversity and NAP% (P < 0.05), but not for Shannon Index or Ambrosia:Artemisia. Precipita- tion was highest in 2013 (462 mm) and lowest in 2012 (301 mm). The role of environmental variables on pollen metrics Overall, the four summative pollen metrics – beta-diver- sity, Shannon index, nonarboreal percentage (NAP%), and Ambrosia:Artemisia – are each partially inﬂuenced by different environmental variables (Table 1). Some envi- ronmental variables (ﬁxed effects) signiﬁcantly inﬂuence more than one of the summative metrics. For example, the area burned within the Flint Hills contributes to both beta-diversity and NAP%. Shannon index is inﬂuenced by bison density. Beta-diversity is inﬂuenced by the area burned within the Flint Hills and the area burned within Konza. NAP% is inﬂuenced by area burned within the Flint Hills and area burned within Konza. Ambrosia:Arte- misia is inﬂuenced by the area burned within 500 m of 5278 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. Table 1. Multiple regression results of the four summative metrics listed at top (beta-diversity, Shannon index, nonarboreal pollen percentage, and Ambrosia:Artemisia), inﬂuenced by the variables listed at the left. Random effects of trap location and year were assessed for signiﬁcance at P < 0.05. Coefﬁcients of the ﬁxed-effect variables that signiﬁcantly contribute to each summative metric are listed. Table 1. Multiple regression results of the four summative metrics listed at top (beta-diversity, Shannon index, nonarboreal pollen percentage, and Ambrosia:Artemisia), inﬂuenced by the variables listed at the left. Random effects of trap location and year were assessed for signiﬁcance at P < 0.05. Coefﬁcients of the ﬁxed-effect variables that signiﬁcantly contribute to each summative metric are listed. Summative pollen metrics Beta-diversity Shannon NAP% Ambrosia:Artemisia Fixed effects Elevation – – – – BareSoil – – – – LimestoneRocks – – – – BisonManure – – – – Bison Density – 0.25 – – Area Burned 500 m – – – –0.14* Area Burned Flint Hills 0.45* – 0.13 – Fire Return Interval – – – – Mean Fire Return Inter. – – – – FireFrequency – – – – TimeSinceLastFire – – – – Area Burned Konza 0.29* – 0.16* – Random effects Trap Not signiﬁcant Signiﬁcant (P<0.001) Signiﬁcant (P<0.001) Signiﬁcant (P<0.001) Year Not signiﬁcant Signiﬁcant (P<0.05) Not signiﬁcant Not signiﬁcant Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.05; Indicates the ﬁxed-effect variable is signiﬁcant at P < 0.01; **Indicates the ﬁxed- effect variable is signiﬁcant at P < 0.001. Squared-chord Distance dissimilarity Squared-chord distance was examined between each sam- ple and all other samples from all traps in all years as an estimation of overall dissimilarity between the multivari- ate assemblages. The spatial differences in the average dis- similarity between each sample and all other samples across Konza (Fig. 4) are generally higher than the tem- poral differences for a single trap among the 4 years (Fig. 5). Thus, within-trap dissimilarity (Fig. 5) is lower than a sample’s average dissimilarity to all other samples (across Konza from all 4 years) (Fig. 4). The average dis- similarity between all samples is 0.38, while the within- trap average dissimilarity is 0.31. Within-trap dissimilarity is lowest from 2011 to 2012 (average 0.28) and highest from 2012 to 2013 (average 0.33). However, some traps have relatively low within-trap dissimilarity, while others have high within-trap dissimilarity (from year to year). For example, Trap 13 has the lowest within-trap dissimi- larity (average 0.11), while Trap 15 has the highest (aver- age 0.70). Minimum dissimilarity for a given sample (the lowest squared-chord distance value of all pairwise com- parisons for a given sample) ranges between 0.01 and 0.30 (Fig. 6), and averages 0.11. The negative inﬂuence of bison density on Shannon index is somewhat puzzling (Table 1), as previous work has suggested that bison grazing increases local plant diversity (Hartnett et al. 1996). This could be explained by over-representation of high pollen producers such as Ambrosia and Amaranthaceae in the pollen assemblage, or, under-representation of low pollen producers such as sunﬂowers (Asteraceae Undiff.) or Fabaceae family plants 5279 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. Figure 3. Kruskal–Wallis ANOVA results by year of the four summative metrics, compared to growing season precipitation. Letters (A, B, C, etc.) indicate groupings from the Kruskal– Wallis pairwise comparisons. Ambrosia: Artemisia showed no signiﬁcant difference among the four years of data and have no letters. The blue shading signiﬁes the high precipitation in the 2013 growing season. From top to bottom: Nonarboreal percentage, Ambrosia:Artemisia, Beta-diversity (SD units), Shannon index, and growing season precipitation (mm). 5280 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 5280 ª 2016 Th Figure 3. Kruskal–Wallis ANOVA results by year of the four summative metrics, compared to growing season precipitation. Figure 3. Kruskal–Wallis ANOVA results by year of the four summative metrics, compared to growing season precipitation. Letters (A, B, C, etc.) indicate groupings from the Kruskal– Wallis pairwise comparisons. Ambrosia: Artemisia showed no signiﬁcant difference among the four years of data and have no letters. The blue shading signiﬁes the high precipitation in the 2013 growing season. From top to bottom: Nonarboreal percentage, Ambrosia:Artemisia, Beta-diversity (SD units), Shannon index, and growing season precipitation (mm). ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Squared-chord Distance dissimilarity Letters (A, B, C, etc.) indicate groupings from the Kruskal– Wallis pairwise comparisons. Ambrosia: Artemisia showed no signiﬁcant difference among the four years of data and have no letters. The blue shading signiﬁes the high precipitation in the 2013 growing season. From top to bottom: Nonarboreal percentage, Ambrosia:Artemisia, Beta-diversity (SD units), Shannon index, and growing season precipitation (mm). 5280 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. Figure 4. Average squared-chord distance dissimilarity between a trap sample and all other samples (from all traps and all years). Green values represent lower average dissimilarity, and red values represent higher average dissimilarity. Figure 4. Average squared-chord distance dissimilarity between a trap sample and all other samples (from all traps and all years). Green values represent lower average dissimilarity, and red values represent higher average dissimilarity. (Amorpha sp., Baptisia sp., or Lespedeza sp.) (Commer- ford et al. 2013). All are common forbs at Konza (Towne 2002). Furthermore, the presence of bison manure (used as a qualitative measure of bison presence/absence) had (Amorpha sp., Baptisia sp., or Lespedeza sp.) (Commer- ford et al. 2013). All are common forbs at Konza (Towne 2002). Furthermore, the presence of bison manure (used as a qualitative measure of bison presence/absence) had no inﬂuence. This suggests that bison herbivory does have an effect on grassland pollen assemblage diversity, but this signal can only be detected with quantitative herbivore abundance. 5281 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. Figure 5. Within-trap squared-chord distance dissimilarity from 2011 to 2012, 2012 to 2013, and 2013 to 2014. Green values represent lowe average dissimilarity, and red values represent higher average dissimilarity. Figure 5. Within-trap squared-chord distance dissimilarity from 2011 to 2012, 2012 to 2013, and 2013 to 2014. Green values represent lower average dissimilarity, and red values represent higher average dissimilarity. Area burned within 500 m of the trap is the only envi- ronmental variable that contributed signiﬁcantly to Ambrosia:Artemisia (negative relationship). Higher levels of Ambrosia pollen (or lower levels of Artemisia, leading to a high ratio) correspond with less area burned. This suggests that grassland ﬁres, at least in a tallgrass prairie like at Konza, may help keep Ambrosia abundance or pol- len productivity low. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Squared-chord Distance dissimilarity Although beta-diversity is inﬂuenced by two broader- scale spatial variables – the area burned within the Flint Hills and the area burned within Konza (Table 1) – this could be partially due to the nature of how the beta- diversity metric is calculated. Here, we calculated tempo- ral beta-diversity, in which the value for each sample was the ratio between the sample diversity for 1 year com- pared to the sample diversity at that same trap for the other 3 years. Because the two above-mentioned variables are equal across all traps for a given year, it is logical that they would have some inﬂuence on the temporal beta- diversity in this case. However, the lack of inﬂuence of the very local, spatial ﬁre variable – area burned within 500 m – is more surprising. The area burned within Konza and the area burned within the Flint Hills operate at a broader spatial scales, indicating that temporal beta- diversity in our pollen samples is more inﬂuenced by annual changes in ﬁres occurring at the local or regional scale than at the very local scale (within 500 m). The inﬂuence of regional burning could be manifested directly through ﬁre driving vegetation composition and structure or could suggest that an indirect regional mechanism, such as prevailing wind direction or speed, is inﬂuencing pollen dispersal (Davis 2000). Additionally, the other four ﬁre variables – the amount of time since the last ﬁre, ﬁre frequency, mean ﬁre return interval, and ﬁre return inter- val – had no inﬂuence. These variables describe temporal aspects of the ﬁre histories at Konza. This ﬁnding is simi- lar to that from Leys et al. (2015), who examined char- coal pieces from these same traps and found that the amount of charcoal was signiﬁcantly inﬂuenced by the area burned within the Flint Hills and Konza, but not by any other ﬁre variables. Additionally, the random effects of trap location and year are signiﬁcant in some of the summative metrics and could be causing the environmental variables (ﬁxed effects) to appear to less signiﬁcant. The random effect of trap location is signiﬁcant in Shannon Index, NAP%, and Ambrosia:Artemisia (Table 1). This signiﬁcance indicates that temporal autocorrelation is likely occurring among the 4 years of pollen data within a given trap. This is not completely surprising, as our SCD values within a trap (Fig. Squared-chord Distance dissimilarity This could occur either by ﬁres ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. 5282 High Dissimilarity With in Grassland Pollen J. L. Commerford et al. Figure 6. Minimum squared-chord distance dissimilarity between a trap sample and all other samples (from all traps and all years). Green value represent lower minimum dissimilarity, and red values represent higher minimum dissimilarity. Please note that the color breaks in the legend are different from the breaks used on Figures 4 and 5. Figure 6. Minimum squared-chord distance dissimilarity between a trap sample and all other samples (from all traps and all years). Green values represent lower minimum dissimilarity, and red values represent higher minimum dissimilarity. Please note that the color breaks in the legend are different from the breaks used on Figures 4 and 5. directly inhibiting Ambrosia growth (Abrams and Gibson 1991), or by promoting the growth of grasses and other forbs (including Artemisia) in place of Ambrosia (Knapp and Seastedt 1986). Additionally, a negative relationship between Ambrosia pollen and charcoal was found over the Holocene in the northern Great Plains (Grimm et al. 5283 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. 2011), suggesting that the Ambrosia-ﬁre relationship is robust across different temporal scales. support greater vegetation species diversity than lowland soils at Konza (Gibson and Hulbert 1987). Additionally, the lower elevations at Konza contain riparian areas with a greater abundance of arboreal vegetation compared to the upland areas dominated by grass and forbs (Veach et al. 2014). Further, arboreal species tend to be rela- tively high pollen producers (Brostrom et al. 2008). Thus, the pollen signal of these woody species, particu- larly Quercus macrocarpa and Quercus muehlenbergii at Konza, could be ampliﬁed in riparian areas where they are already more abundant on the landscape, muting the assemblage diversity assessed by the Shannon Index. It is important to reiterate that the inﬂuence of elevation is not statistically signiﬁcant within the 4-year record of pollen data presented here. However, additional years of data from these traps could reveal or refute the inﬂu- ence of local elevation differences on these pollen assem- blages. Squared-chord Distance dissimilarity 5) were also found to be lower than the SCD values across all traps and years (Fig. 4), indicating higher simi- larity among the pollen assemblages at a given trap. Vege- tation composition is known for being the strongest driver of pollen assemblage composition. While we did not measure vegetation composition as a driver of pollen assemblages, its relationship has been quantiﬁed with pol- len assemblages in various biomes. At Konza, vegetation varies less at a given trap from year to year and more from trap to trap, so it is logical that pollen assemblages would also vary less at a given trap from year to year. In addition, the random effect of year is also signiﬁcant in Shannon Index, indicating spatial autocorrelation in Shannon index values (but not in NAP%, beta-diversity, or Ambrosia:Artemisia values) across traps within a given year. This suggests that an annual-scale driver not included in the regression analyses is inﬂuencing Shannon index. Although there is little topographic relief (<100 m) in the study area compared to mountain environments, ele- vation changes might affect pollen spectra. While eleva- tion did not signiﬁcantly inﬂuence any of the summative metrics at P < 0.05, it was very close to con- tributing to NAP% (P = 0.057, negative coefﬁcient) and Shannon Index (P = 0.074, positive coefﬁcient), and so is worth mentioning. In a montane region in Norway, pollen assemblages from a set of traps spanning a larger elevational transect (~500 m) were found to vary greatly in their composition, mostly due to differences in vege- tation composition and structure (Birks and Bjune 2010; Bjune 2014). Although the Konza traps span a smaller elevational gradient, the inﬂuence of elevation on pollen assemblages here could also be due to systematic differ- ences in vegetation at lower versus higher elevations, although the inﬂuence is not signiﬁcant in the 4-year record here. Yet, upland soils have been found to High Dissimilarity With in Grassland Pollen J. L. Commerford et al. High Dissimilarity With in Grassland Pollen assemblages) to go undetected during the time between samples (Liu et al. 2012). Beta-diversity has been calcu- lated on vegetation proxies from lacustrine sediment cores in various regions around the world (Birks 2006; Leys et al. 2014), including pollen from the Great Plains (Commerford et al. 2016), but not on annual modern pollen samples. Our ANOVA results demonstrate statisti- cally signiﬁcant differences in beta-diversity between some of the years from 2011 to 2014 (Fig. 3). Yet, despite these differences, our annual averages for beta-diversity range only between 0.8 and 1.2 SD units. Comparatively, Holo- cene pollen assemblages from Fox Lake in the northern Great Plains had beta-diversity values that averaged around 0.8 SD units (4 SD units would imply full turn- over), suggesting that grassland vegetation at that site exhibited little change in beta-diversity over much of the Holocene, despite recurring ﬁre and drought (Commer- ford et al. 2016). Our beta-diversity values are similar, suggesting that the amount of change is comparable on an annual time scale. In long-term records of modern pollen, an anomalous year of pollen data (either summarized by a single metric or by individual key taxa) could be muted by the other years of data if averaged over the entire record. Alterna- tively, a single anomalous year might signiﬁcantly inﬂu- ence the average if it is substantially different from the other years of data. The role of anomalous individual years could be especially relevant for fossil pollen records from nonlaminated lacustrine sediment cores, where a 1 cm sample often spans a decade or more (depending on the location and the deposition rate; Goring et al. 2012; Minckley et al. 2012a). Ultimately, modern multi- year records that match the time frame of deposition rates in lacustrine cores from the same region could high- light “tipping points” for how anomalous a single year needs to be to affect an average. The longest-running annual Tauber trap pollen records of which we are aware are in Europe, some of which span 8 years or more (H€attestrand 2013; Pidek et al. 2013; Bjune 2014). In the Great Plains, pollen and sediment deposition rates in lakes are relatively rapid (e.g., 5 years cm1, Grimm et al. 2011) and so fewer years of pollen monitoring would be needed to match their temporal resolution. High Dissimilarity With in Grassland Pollen The medical community has monitored pollen taxa on the Great Plains for decades and has found a direct inﬂuence of temperature on pollen production of common allergenic taxa such as Ambrosia and Juniperus (Levetin 2001; Ziska et al. 2011). Although the collection devices used in those studies are speciﬁcally designed to trap airborne pollen rather than deposited pollen – and thus may not serve as a direct comparison to pollen deposited in lake sediment – these records could be sufﬁciently long to demonstrate the impact of a single anomalous year of pollen produc- tion on a multiyear average. The pollen data from 2013 are the most extreme of our 4-year record: lowest beta-diversity, lowest Shannon diversity, highest Ambrosia:Artemisia, and highest NAP% (Fig. 3). This change in the average for all traps in 2013 seems to indicate a regional-scale driver. Higher precipita- tion in the 2013 growing season (462.1 mm from May to August, an increase of at least 35% over the other 3 years) is a likely cause. The mechanism could be either through a direct inﬂuence on pollen production of certain nonarboreal taxa (McLauchlan et al. 2011) or through increased inwash of pollen from overland ﬂow (Birks and Bjune 2010; Bjune 2014). High summer precipitation has been associated with high levels of Ambrosia pollen, while high spring precipitation has been associated with high levels of Poaceae pollen at other monitoring sites in the region (McLauchlan et al. 2011). Given that Ambrosia and Poaceae are the two most abundant nonarboreal pol- len types in our 4-year dataset, an increase in their pro- duction would certainly affect our summative metrics. Ambrosia and Poaceae abundances are highest in 2013 (compared to all other years) at the majority of the traps (Fig. 2), providing further support for the increase being due to production. Higher ﬂowering is a possible instiga- tor of this increase in production (Hicks 2006). However, increased inwash of pollen from overland ﬂow cannot be ruled out as another potential cause. Birks and Bjune (2010) identiﬁed inwash as a source of plant macrore- mains in traps in a Norwegian woodland, and Bjune (2014) also suggested it as a mechanism for pollen depo- sition. Given the topography of the limestone and mud- stone hills at Konza, some of our traps at the lower elevations could be particularly susceptible to pollen deposition via inwash. Annual differences in pollen assemblage diversity In lacustrine sediment cores, pollen sampling for vegeta- tion reconstruction often does not follow a continuous scheme. Therefore, there is the potential for composi- tional changes in vegetation (inferred from pollen 5284 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High dissimilarity among samples The limited temporal resolution of nonlaminated lacus- trine sediment cores has been seen as a potential issue for accurately reconstructing vegetation from pollen assem- blages throughout the Holocene (Hicks and Hyv€arinen 1999), because it is impossible to achieve an annual-scale sampling resolution. However, even in the best case sce- nario, it is likely that sedimentary pollen analysis will not achieve ﬁner than subdecadal resolution (Joosten and de Klerk 2007). Yet, the results of our squared-chord dis- tance dissimilarity analysis indicate that pollen assemblage composition is more dissimilar across all traps and years (Fig. 4), than it is between years at a single trap (Fig. 5). The relatively lower within-trap dissimilarity values (year to year) (Fig. 5) indicate that, although signiﬁcant, annual-scale differences in pollen productivity or subtle annual changes in grassland vegetation composition may 5285 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. High Dissimilarity With in Grassland Pollen J. L. Commerford et al. not have as much of an impact on pollen assemblage composition as spatial differences. compared to samples from soils or lake sediments. Thus, the mean abundance values from all of the traps would likely be most useful when comparing our samples to grassland fossil pollen records in future work (as sug- gested by Bjune 2014). Our pollen samples from the Konza prairie have an overall average dissimilarity of 0.38 (the average of the SCD values between all pairs of samples), but an average minimum dissimilarity of 0.11 (the average of the lowest SCD value for all samples). This difference between the overall average and the average minimum dissimilarity between samples indicates that most samples have a close compositional match, but also are quite dissimilar from many of the other samples in our dataset. Our results are similar to those from modern pollen samples from other grassland regions of North America and Africa. In Africa, modern grassland pollen samples were found to have an average minimum dissimilarity of 0.13, while savanna samples were slightly higher at 0.16 (Gajewski et al. 2002). Our Konza prairie samples exhibited slightly lower average minimum dissimilarity (0.11). In Oklahoma and north-central Texas, tallgrass prairie pollen samples from Tauber traps have also been found to be fairly similar to each other (0.10), but dissimilar to samples from short grass and mixed grass prairie in west Texas and New Mexico (0.40) (Hoyt 2000). Conclusion Modern tallgrass prairie pollen assemblages are more simi- lar temporally than spatially, as shown by SCD values across the 4-year record. Our SCD values similar to SCD values for pollen samples from modern African grasslands, but higher compared to other modern grasslands in North America. The higher dissimilarity compared to other North American grasslands’ pollen samples could be due to our larger sample size, and the deliberate placement of the traps in this study to include a range of elevations, burning frequencies, and grazing intensities. In addition, annual differences in pollen composition and diversity are signiﬁ- cant in three of the four summative metrics that we exam- ined: beta-diversity, Shannon index, and nonarboreal pollen abundance (not signiﬁcant in Ambrosia:Artemisia). Interannual variability in precipitation is signiﬁcantly related to these differences. The 2013 growing season expe- rienced signiﬁcantly higher precipitation than 2011, 2012, and 2014, as well as the highest NAP%, lowest Shannon Index, and lowest beta-diversity of the 4-year record. Vari- ables of topography, ﬁre, and grazing do have some effect on the diversity and composition of pollen taxa among traps, although their individual signiﬁcance varies depend- ing on the metric. Further long-term modern pollen records from grasslands are essential in order to better establish the degree of variation in pollen production and deposition on an annual basis. All of the traps in this study are located within a 3487 hectare area that is predominantly grassland despite some local differences in the amount and type of nearby woody vegetation. Yet, the pollen assemblages from the 27 traps would not all be considered analogs for each other using the Modern Analog Technique with a suggested SCD threshold of 0.20–0.30 (Williams and Shuman 2008). The high variation in these modern pollen assemblages implies that deﬁning a pollen assemblage as “grassland” (particu- larly when examining fossil pollen assemblages) could be more difﬁcult than previously thought. However, Tauber traps typically detect a very local vegetation signal given their relatively small 5-cm opening compared to lake sur- faces, which have a much larger source area (Calcote 1995; Sugita 2007b). In a lake, the pollen from the entire source area would be mixed together (Sugita 2007a) and so the small source area associated with traps may be a reason for the high dissimilarity among our samples High dissimilarity among samples Among our Konza prairie samples, the average dissimilarity between all samples is 0.38, which is more comparable to the 0.40 dissimilarity found between the different grassland types by Hoyt (2000). Our samples could be more dissimilar because our traps were intentionally placed to span a range of dif- ferent burning and grazing treatments to examine the inﬂuence of those factors on the diversity and composi- tion of the pollen assemblages. Hoyt (2000) aimed to identify pollen signatures among different grassland types (tallgrass, mixed grass, shortgrass) and therefore did not discuss any variation in burning and grazing, if any, among sample sites. References Abrams, M.D., and D.J.Gibson . 1991. 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Courtois for photographic assistance. We thank Simon Goring and an anonymous reviewer for providing helpful comments that improved an earlier version of this manu- script. Funding to maintain the ﬁre and grazing treat- ments at the Konza Prairie LTER was National Science Foundation DEB-0823341. JC was funded by National Science Foundation BCS-0955225 to KM, a GK-12 fellow- ship (National Science Foundation DGE-0841414 to C. Ferguson), and an National Science Foundation Doctoral Dissertation Research Improvement Grant (NSF BCS- 1558228 to KM and JC). 5286 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. J. L. Commerford et al. High Dissimilarity With in Grassland Pollen Conﬂict of Interest climatic ﬂuctuations during the Holocene at Fox Lake, Minnesota (USA). Holocene 26:302–313. Minnesota (USA). Holocene 26:302–313. None declared. Cook, E. R., R. Seager, R. R. Jr Heim, R. S. Vose, C. Herweijer, and C. Woodhouse. 2010. 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Pollen representation of vegetation in quaternary sediments: theory and method in patchy vegetation. J. Ecol. 82:881–897. Weng, C., H. Hooghiemstra, and J. F. Duivenvoorden. 2007. Response of pollen diversity to the climate-driven altitudinal shift of vegetation in the Colombian Andes. Phil. Trans. R. Soc. B: Biol. Sci. 362:253–262. Sugita, S. 2007a. Theory of quantitative reconstruction of pollen I: pollen from large lakes REVEALS regional vegetation composition. Holocene 17:229–241. Sugita, S. 2007b. Theory of quantitative reconstruction of pollen II: all you need is LOVE. Holocene 17:243–257. Wickham, H. 2016. Scales: scale functions for visualization. Open-access R package, Wickham. https://cran.r- project.org/web/packages/scales/index.html. Sugita, S., T. Parshall, and R. Calcote. 2006. Detecting differences in vegetation among paired sites using pollen records. Holocene 16:1123–1135. project.org/web/packages/scales/index.html. Williams, J. W., and B. Shuman. 2008. Obtaining accurate and precise environmental reconstructions from the modern analog technique and North American surface pollen dataset. Quatern. Sci. Rev. 27:669–687. Tauber, H. 1974. Static nonoverload pollen collector. New Phytologist 73:359–369. Williams, J. W., B. Shuman, and P. J. Bartlein. 2009. Rapid responses of the prairie-forest ecotone to early Holocene aridity in mid-continental North America. Global Planet. Change 66:195–207. Ter Braak, C. J. F. 1986. Canonical correspondence analysis a new eigenvector technique for multivariate direct gradient analysis. Ecology (Washington DC) 67:1167–1179. Ter Braak, C.J.F., and P. Smilauer (2012). Canoco reference manual and user’s guide: software for ordination, version 5.0. Microcomputer Power, Ithaca, NY. Ziska, L., K. Knowlton, C. Rogers, D. Dalan, N. Tierney, M. A. Elder, et al. 2011. Recent warming by latitude associated with increased length of ragweed pollen season in central North America. Proc. Natl Acad. Sci. 108:4248– 4251. Tonello, M. S., and A. R. Prieto. 2008. Modern vegetation- pollen-climate relationships for the Pampa grasslands of Argentina. J. Biogeogr. 35:926–938. associated with increased length of ragweed pollen season in central North America. Proc. Natl Acad. Sci. 108:4248– 4251. Towne, E. G. 2002. References Vascular plants of Konza Prairie Biological Station: an annotated checklist of species in a Kansas tallgrass prairie. Sida 20:269–294. ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd. Supporting Information Additional Supporting Information may be found online in the supporting information tab for this article: Umbanhowar, C. E., P. Camill, C. E. Geiss, and R. Teed. 2006. Asymmetric vegetation responses to mid-Holocene aridity at the prairie–forest ecotone in south-central Minnesota. Quatern. Res. 66:53–66. Appendix S1. R code for multiple regressions with ﬁxed and random effects, ANOVAs, and squared-chord dis- tance calculations. Veach, A. M., W. A. Dodds, and A. Skibbe. 2014. Fire and grazing inﬂuences on rates of riparian woody 5289 ª 2016 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.
https://openalex.org/W4361892177	https://figshare.com/articles/journal_contribution/Supplementary_Figure_Legends_1-2_from_The_p38_MAPK_Pathway_Mediates_Aryl_Propionic_Acid_Induced_Messenger_RNA_Stability_of_p75_sup_NTR_sup_in_Prostate_Cancer_Cells/22371003/1/files/39816090.pdf	English	null	Supplementary Figure Legends 1-2 from The p38 MAPK Pathway Mediates Aryl Propionic Acid–Induced Messenger RNA Stability of p75<sup>NTR</sup> in Prostate Cancer Cells	null	2,023	cc-by	433	Supplementary Figure Legends Supplementary Figure S1. A, p75NTR mRNA and protein levels following ibuprofen treatment. LNCaP cells were treated with 0 to 2 mM ibuprofen for 24 hours. Cell lysates were collected and equal amounts of protein were subjected to SDS-PAGE and immunoblot analysis using an antibody to p75NTR or β-actin (β-act) for the loading control. Alternatively, RNA was isolated from LNCaP cells following 24 hour ibuprofen treatment and p75NTR and GAPDH mRNA levels were determined by RT-PCR. B, time course induction of p75NTR mRNA levels. LNCaP cells were treated with 2 mM ibuprofen for 0, 2, 4, 8, or 24 hours. RNA was isolated at each time point and levels of p75NTR and GAPDH were determined by RT-PCR. C and D, increase in p75NTR mRNA stability following ibuprofen treatment. LNCaP cells were treated with 2 mM ibuprofen (IBU) or DMSO vehicle control for 8 hours followed by the addition of 5 µg/mL Actinomycin D (ActD). RNA was isolated at 0, 2, 4, 8, and 12 hours following the addition of ActD, and p75NTR mRNA levels were determined by RT-PCR. The intensity of the bands for p75NTR were analyzed by densitometry and graphed as a percentage of the 0 hour time point. Supplementary Figure S2. A, activation of the p38 MAPK pathway by ibuprofen. LNCaP cells were treated with 2 mM ibuprofen for 0, 5 minutes, 1 hour, 4 hours, or 8 hours. Cell lysates were collected and equal amounts of protein were subjected to SDS-PAGE and immunoblot analysis using antibodies to 2 2 phosphorylated p38 MAPK (p-p38) or phosphorylated MK2 (p-MK2). Blots for p-p38 and p-MK2 were stripped and reprobed for total p38 MAPK and total MK2, respectively. β-actin (β-act) was used as the loading control. B, validation assay for siRNA knockdown of p38α, MK2, MK3, and HuR. LNCaP cells were transfected for 72 hours with non-targeting siRNA or siRNA targeting p38α, MK2 and MK3, or HuR. Cell lysates were collected and efficacy of siRNA knockdown was determined by SDS-PAGE and immunoblot analysis using antibodies to p38α, MK2, MK3, or HuR. β-actin (β-act) was used as the loading control. C, knockdown of p38 MAPK, MK2 and MK3, or HuR prevents induction of p75NTR by ibuprofen. LNCaP cells were transfected with non- targeting siRNA, siRNA for p38α, siRNAs for MK2 and MK3, or siRNA for HuR for 72 hours. Supplementary Figure Legends Following transfection, cells were treated with 2 mM ibuprofen (IBU) or DMSO vehicle control (CON) for 24 hours. Cell lysates were collected and expression of p75NTR was determined by SDS-PAGE and immunoblot analysis. β-actin (β-act) was used as the loading control.
https://openalex.org/W3125527790	https://www.repository.cam.ac.uk/bitstreams/5d7403ba-147c-4f24-bb4a-6e1f1b776013/download	English	null	Multioscillating black holes	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	19,733	Published for SISSA by Springer Received: February 9, 2021 Accepted: March 24, 2021 Published: May 3, 2021 Multioscillating black holes JHEP05(2021)011 Open Access, c⃝The Authors. Article funded by SCOAP3. Takaaki Ishii,a Keiju Murata,b Jorge E. Santosc and Benson Wayd https://doi.org/10.1007/JHEP05(2021)011 Contents 1 Introduction 1 2 Hypersphere isometries and Wigner D-matrices 3 3 Cohomogeneity-1 spacetime with rotating scalar ﬁeld 5 4 Geons and superradiant instability of Myers-Perry-AdS 9 5 Black resonators 11 6 Hairy Myers-Perry-AdS and boson stars 12 7 Hairy black resonator and graviboson star 14 8 Phase diagram 16 9 Phase diagram for higher j 17 10 Conclusion 18 A Technical details 21 A.1 Equations of motion 21 A.2 Boundary conditions at inﬁnity 22 A.3 Boundary conditions at the origin for horizonless solutions 23 A.4 Boundary conditions at the horizon 24 A.5 Physical quantities 25 A.6 Interpolation of hairy black resonator data 26 B Instability of hairy MPAdS 27 C Perturbative solution of boson star 28 C.1 Higher order perturbation 28 C.2 Large-j expansion of the perturbative solution 30 Contents 1 Introduction 1 2 Hypersphere isometries and Wigner D-matrices 3 3 Cohomogeneity-1 spacetime with rotating scalar ﬁeld 5 4 Geons and superradiant instability of Myers-Perry-AdS 9 5 Black resonators 11 6 Hairy Myers-Perry-AdS and boson stars 12 7 Hairy black resonator and graviboson star 14 8 Phase diagram 16 9 Phase diagram for higher j 17 10 Conclusion 18 A Technical details 21 A.1 Equations of motion 21 A.2 Boundary conditions at inﬁnity 22 A.3 Boundary conditions at the origin for horizonless solutions 23 A.4 Boundary conditions at the horizon 24 A.5 Physical quantities 25 A.6 Interpolation of hairy black resonator data 26 B Instability of hairy MPAdS 27 C Perturbative solution of boson star 28 C.1 Higher order perturbation 28 C.2 Large-j expansion of the perturbative solution 30 JHEP05(2021)011 Takaaki Ishii,a Keiju Murata,b Jorge E. Santosc and Benson Wayd aDepartment of Physics, Kyoto University, Kitashirakawa Oiwake-cho, Kyoto 606-8502, Japan bDepartment of Physics, College of Humanities and Sciences, Nihon University, Sakurajosui, Tokyo 156-8550, Japan cDAMTP, Centre for Mathematical Sciences, University of Cambridge, Wilberforce Road, Cambridge CB3 0WA, U.K. dDepartament de Física Quàntica i Astrofísica, Institut de Ciències del Cosmos, Universitat de Barcelona, Martí i Franquès, 1, E-08028 Barcelona, Spain E-mail: ishiitk@gauge.scphys.kyoto-u.ac.jp, murata.keiju@nihon-u.ac.jp, jss55@cam.ac.uk, benson@icc.ub.edu Abstract: We study rotating global AdS solutions in ﬁve-dimensional Einstein gravity coupled to a multiplet complex scalar within a cohomogeneity-1 ansatz. The onset of the gravitational and scalar ﬁeld superradiant instabilities of the Myers-Perry-AdS black hole mark bifurcation points to black resonators and hairy Myers-Perry-AdS black holes, respectively. These solutions are subject to the other (gravitational or scalar) instability, and result in hairy black resonators which contain both gravitational and scalar hair. The hairy black resonators have smooth zero-horizon limits that we call graviboson stars. In the hairy black resonator and graviboson solutions, multiple scalar components with diﬀerent frequencies are excited, and hence these are multioscillating solutions. The phase structure of the solutions are examined in the microcanonical ensemble, i.e. at ﬁxed energy and angular momenta. It is found that the entropy of the hairy black resonator is never the largest among them. We also ﬁnd that hairy black holes with higher scalar wavenumbers are entropically dominant and occupy more of phase space than those of lower wavenumbers. Keywords: Black Holes, AdS-CFT Correspondence Keywords: Black Holes, AdS-CFT Correspondence ArXiv ePrint: 2101.06325 Open Access, c⃝The Authors. Article funded by SCOAP3. Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction Through superradiant scattering, energy can be extracted from rapidly rotating black holes (see [1] for a review). In global anti-de Sitter space (AdS), the reﬂecting boundary causes these black holes to be unstable to the superradiant instability [2–11], whose ultimate endpoint remains an open problem [12–15]. Because small black holes typically have – 1 – high angular frequency, the ultimate conﬁguration of low-energy states in AdS likewise remains unknown. Early work on this problem involved studying perturbations of the Kerr-AdS and Myers-Perry-AdS black holes (the higher-dimensional analog of Kerr-AdS [16–19], see [20] for a review), where quasi-normal spectra were obtained in [10, 11]. For speciﬁc modes, the onset of the superradiant instability occurs at a frequency where ℑ(ω) = 0, but ℜ(ω) ̸= 0. This suggests the existence of a time-periodic black hole with a single helical Killing vector that branches from these onsets. Such black holes, called black resonators, which can be viewed as black holes with a gravitational hair, were later constructed in [13], where it was found that they have higher entropy (horizon area) than the corresponding Kerr-AdS black hole with the same mass and angular momentum. JHEP05(2021)011 It is therefore entropically permissible for Kerr-AdS black holes to evolve towards black resonators. However, though black resonators are stable to the mode that generated them, they are still rapidly rotating and hence remain unstable to other, typically higher, superradiant modes [12–15, 21]. It appears, therefore, that the instability leads to a cascade with higher and higher modes growing in time. If there is indeed an unceasing energy cascade towards higher modes, there will eventually be a signiﬁcant amount of energy placed in sub-Planckian length scales, which can be viewed as a violation of the weak cosmic censorship conjecture [22]. To date, there is only a single study of time-evolution involving the rotational super- radiance of AdS [15]. In Kerr-AdS, there is typically one unstable mode that dominates the dynamics at early times. Evolution then proceeds towards a black resonator until the instabilities of the black resonator itself begin to take over and drive the continuing evolution. The evolution in [15] was not continued further due to numerical limitations. Because of the lack of symmetries and the long time-scales involved, the study of the superradiant instability is a signiﬁcant numerical challenge. It is therefore fortunate that a simpliﬁcation in a more limited setting has been found. 1For the superradiant instability of an electromagnetic perturbation isolated from these modes, a cohomogeneity-1 photonic black resonator was also constructed in [25]. 1 Introduction By moving to ﬁve dimensions and allowing both angular momenta to be equal, black resonators with a cohomogeneity-1 ansatz (i.e. the metric functions depend only on a single variable, and the solution can be obtained by solving ODEs) were constructed in [23]. The scalar, electromagnetic, and gravitational quasinormal modes of these black resonators were studied shortly thereafter in [24].1 As anticipated, black resonators are unstable to higher modes. These higher-mode instabilities of black resonators also have their individual onsets, from which new black resonators having multiple frequencies could be generated. We will refer such solutions as multi black resonators. Here, we set out to construct such multi black resonator solutions and to study their relationship with black resonators and Myers- Perry-AdS black holes. Ideally, we would study multi black resonators that are generated by gravitational perturbations of black resonators. However, these perturbations break too many symmetries of the original black resonator solution. We therefore focus on multi black resonators that are generated by scalar ﬁelds. Ordinarily, a scalar ﬁeld would also – 2 – break most of these symmetries, but we will rely on a multiplet scalar constructed using Wigner D-matrices, from which the cohomogeneity-1 structure can be preserved. A scalar doublet version of such a system was previously studied in [9, 26].2 The higher scalar multiplets we introduce can coexist with oscillations of the metric, but do not contribute additional extra oscillating frequencies to the metric. Even in this limited setting, the full space of solutions is intricate. There are Myers- Perry-AdS black holes, black holes with scalar hair, black resonators, and now hairy black resonators, which are a kind of multi black resonator. In addition, there are boson stars, geons, and now graviboson stars, which are all horizonless solutions that serve as the zero-size limit of hairy Myers-Perry-AdS, black resonators, and hairy black resonators, respectively. All of these solutions compete thermodynamically when they share the same energy and angular momenta. We will compute the full phase diagram of this system. Perhaps surprisingly, we ﬁnd that hairy black resonators are never dominant in such a phase diagram. JHEP05(2021)011 Another advantage of the multiplet scalar model is that diﬀerent nonlinear solutions generated by diﬀerent mode-instabilities can be consistently compared with one another, while maintaining a cohomogeneity-1 ansatz. 1 Introduction This will allow us to show that black holes generated from higher scalar mode instabilities have higher entropy and occupy a larger region of phase space than those from lower modes. Similar conclusions reached by previous work were only argued by extrapolating perturbative calculations, and here we are able to perform full nonlinear calculations and compute the actual phase boundaries. This paper is structured as follows. In the next section, we review some basic prop- erties of isometries of S3 and Wigner D-matrices that we use in the construction of our ansatz. Then in section 3, we describe details of our ansatz. Sections 4 and 5 review the Myers-Perry-AdS solution, geons, and black resonators in this ansatz, which were studied in [23, 24]. Section 6 discusses hairy black holes and boson stars which are higher multi- plet versions of those in [9]. Then, in section 7, we present the entirely new hairy black resonator and graviboson star solutions. The entire phase diagram of all solutions then pieced together in section 8, and then we compare the results to a higher wavenumber in section 9. We then ﬁnish with some concluding remarks in section 10. The appendix contains technical details. 2See also [27, 28] for a study covering diﬀerent number of dimensions. 2 Hypersphere isometries and Wigner D-matrices Our metric ansatz will contain deformations of an S3 whose perturbations can naturally be written in terms of Wigner D-matrices Dj mk(θ, φ, χ). Let us therefore begin with a review of the Wigner D-matrices, which we will later use for designing a cohomogeneity-1 scalar ﬁeld ansatz. (See refs. [7, 24, 29–33] for an introduction to the Wigner D-matrix and its applications in gravitational perturbation theory.) We focus on the SO(4) ≃SU(2)L × SU(2)R isometry of S3, whose metric can be written as 1 dΩ2 3 = 1 4(σ2 1 + σ2 2 + σ2 3) , (2.1) (2.1) – 3 – where σi (i = 1, 2, 3) are 1-forms deﬁned by where σi (i = 1, 2, 3) are 1-forms deﬁned by where σi (i = 1, 2, 3) are 1-forms deﬁned by where σi (i = 1, 2, 3) are 1-forms deﬁned by σ1 = −sin χdθ + cos χ sin θdφ , σ2 = cos χdθ + sin χ sin θdφ , σ3 = dχ + cos θdφ . (2.2) σ1 = −sin χdθ + cos χ sin θdφ , (2.2) σ2 = cos χdθ + sin χ sin θdφ , (2.2) These satisfy the SU(2) Maurer-Cartan equation dσi = (1/2)ϵijkσj ∧σk. The coordinate ranges are 0 ≤θ < π, 0 ≤φ < 2π, and 0 ≤χ < 4π, and have a twisted periodicity (θ, φ, χ) ≃(θ, φ + 2π, χ + 2π) ≃(θ, φ, χ + 4π). The Killing vectors generating SU(2)L and SU(2)R, denoted by ξi and ¯ξi, respectively, are given by JHEP05(2021)011 ξ1 = cos φ∂θ + sin φ sin θ ∂χ −cot θ sin φ∂φ , ξ2 = −sin φ∂θ + cos φ sin θ ∂χ −cot θ cos φ∂φ , (2.3) (2.3) ξ3 = ∂φ , and and ¯ξ1 = −sin χ∂θ + cos χ sin θ ∂φ −cot θ cos χ∂χ , ¯ξ2 = cos χ∂θ + sin χ sin θ ∂φ −cot θ sin χ∂χ , ¯ξ3 = ∂χ . (2.4) (2.4) Note that ¯ξi are the dual vectors of σi: (σi)α(¯ξj)α = δij (α = θ, φ, χ). Note that ¯ξi are the dual vectors of σi: (σi)α(¯ξj)α = δij (α = θ, φ, χ). 2 Hypersphere isometries and Wigner D-matrices Using language from quantum mechanics, we can deﬁne the “angular momentum” operators Using language from quantum mechanics, we can deﬁne the “angular momentum” erators Li = iξi , Ri = i¯ξi , (2.5) (2.5) which satisfy the commutation relations [Li, Lj] = iϵijkLk and [Ri, Rj] = −iϵijkRk. These operators are Hermitian under the inner product on the S3, (f, g) ≡1 8 Z π 0 dθ Z 2π 0 dχ Z 4π 0 dχ sin θf∗(θ, φ, χ)g(θ, φ, χ) . (2.6) (2.6) Under the SU(2)L and SU(2)R, the 1-forms introduced in eq. (2.2) transform as Liσj = 0 , Riσj = −iϵijkσk , (2.7) (2.7) where the operations of Li and Ri are deﬁned by Lie derivatives. From the ﬁrst equa- tion of (2.7), one can see that σi are invariant under SU(2)L. For this reason, they are called SU(2)-invariant 1-forms. The second equation means that Ri generate the three- dimensional rotation of the “vector” (σ1, σ2, σ3). In particular, R3 generates U(1)R ⊂SU(2)R, which corresponds to rotation in the (σ1, σ2)-plane. The invariance of dΩ2 3 under SU(2)L × SU(2)R can be easily checked by using eq. (2.7). The generators Li and Ri share the same Casimir operator: L2 ≡L2 1 + L2 2 + L2 3 = R2 1 + R2 2 + R2 3, and the set of commutative operators is given by (L2, Lz, Rz). The Wigner D-matrix Dj mk(θ, φ, χ) is deﬁned to be the eigenfunction of these operators: L2Dj mk = j(j + 1)Dj mk , LzDj mk = mDj mk , RzDj mk = kDj mk , (2.8) (2.8) – 4 – where the ranges of the quantum numbers (j, m, k) are where the ranges of the quantum numbers (j, m, k) are where the ranges of the quantum numbers (j, m, k) are j = 0, 1/2, 1, 3/2, . . . , m = −j, −j + 1, . . . , j , k = −j, −j + 1, . . . , j . (2.9) (2.9) The Wigner D-matrices are orthogonal under the inner product (2.6), The Wigner D-matrices are orthogonal under the inner product (2.6), (Dj′ m′k′, Dj mk) = 2π2 2j + 1δmm′δkk′δjj′ . 2 Hypersphere isometries and Wigner D-matrices (2.12) is simply written as ⃗D∗ k′ · ⃗Dk = δk′k . (2.14) (2.14) 2 Hypersphere isometries and Wigner D-matrices (2.10) (2.10) JHEP05(2021)011 JHEP05(2021)011 We can also deﬁne the ladder operators L± = Lx ± iLy and R± = Ry ± iRx, which shift the “orbital angular momenta” of Dj mk as We can also deﬁne the ladder operators L± = Lx ± iLy and R± = Ry ± iRx, which shift the “orbital angular momenta” of Dj mk as L+Dj mk = εm+1Dj (m+1)k , L−Dj mk = εmDj (m−1)k , R+Dj mk = ϵk+1Dj m(k+1) , R−Dj mk = ϵkDj m(k−1) , (2.11) (2.11) where εm = p (j + m)(j −m + 1) and ϵk = p (j + k)(j −k + 1). where εm = p (j + m)(j −m + 1) and ϵk = p (j + k)(j −k + 1). The Wigner D-matrices satisfy a convenient formula for summatio where εm = p (j + m)(j −m + 1) and ϵk = p (j + k)(j −k + 1). The Wigner D matrices satisfy a convenient formula for summatio where εm p (j + m)(j m + 1) and ϵk p (j + k)(j k + 1). The Wigner D-matrices satisfy a convenient formula for summation, Wigner D-matrices satisfy a convenient formula for summation, The Wigner D-matrices satisfy a convenient formula for summation, j X m=−j (Dj mk′)∗Dj mk = δk′k . (2.12) (2.12) This can be proved easily using the ladder operators. Using eqs. (2.8) and (2.11), we ﬁnd Li(Pj m=−j(Dj mk′)∗Dj mk) = 0. Hence, the left hand side of (2.12) is a constant. Integrating this equation over the S3 as eq. (2.6) and using eq. (2.10), we ﬁnd that the constant is δkk′. For later convenience we introduce a (2j + 1) component vector ⃗Dk by For later convenience, we introduce a (2j + 1)-component vector ⃗Dk by ⃗Dk =        Dj m=j,k Dj m=j−1,k ... Dj m=−j,k        . (2.13) (2.13) Although ⃗Dk also depends on the index j, we suppress it for notational simplicity because we will generally keep j ﬁxed once the content of the scalar ﬁeld is speciﬁed. In this notation, eq. (2.12) is simply written as Although ⃗Dk also depends on the index j, we suppress it for notational simplicity because we will generally keep j ﬁxed once the content of the scalar ﬁeld is speciﬁed. In this notation, eq. 3 Cohomogeneity-1 spacetime with rotating scalar ﬁeld We now describe the ansatz for resonating cohomogeneity-1 spacetimes. We will show that the energy-momentum tensor of the matter ﬁeld we introduce is consistent with the symmetries of the metric and therefore that the equations of motion reduce to a consistent set of ordinary diﬀerential equations. – 5 – We consider the following ﬁve-dimensional Einstein-scalar system with a negative cos- mological constant: S = 1 16πG5 Z d5x√−g R + 12 L2 −∂µ⃗Π∗· ∂µ⃗Π , (3.1) (3.1) where ⃗Π denotes a (2j +1)-component complex scalar multiplet, G5 is the ﬁve-dimensional Newton constant, and L is the AdS radius. Hereafter, we set L = 1. For the metric, we take the cohomogeneity-1 ansatz [23] where ⃗Π denotes a (2j +1)-component complex scalar multiplet, G5 is the ﬁve-dimensional Newton constant, and L is the AdS radius. Hereafter, we set L = 1. For the metric, we take the cohomogeneity-1 ansatz [23] For the metric, we take the cohomogeneity-1 ansatz [23] ds2 = −(1 + r2)f(r)dτ 2 + dr2 (1 + r2)g(r) + r2 4 α(r)σ2 1 + 1 α(r)σ2 2 + β(r)(σ3 + 2h(r)dτ)2 , (3.2) JHEP05(2021)011 JHEP05(2021)011 (3.2) where σi were deﬁned in (2.2). For the scalar multiplet, we take ⃗Π(τ, r, θ, φ, χ) = X k∈K Φk(r) ⃗Dk(θ, φ, χ) , (3.3) (3.3) where Φk(r) are real scalar ﬁelds, and K is deﬁned by K = {j, j −2, j −4, · · · , −j} (j : integer), K = {j, j −2, j −4, · · · , −j + 1} (j : half integer) . (3.4) (3.4) We ﬁrst comment on the metric ansatz (3.2) before later addressing the scalar. This metric ansatz preserves SU(2)L but breaks SU(2)R. If α(r) = 1, then a U(1)R ⊂SU(2)R symmetry generated by R3 is restored. To see this, we can use the fact that σ2 1 + σ2 2 = dθ2 + sin2 θdφ2 = dΩ2 2 , (3.5) (3.5) which is independent of χ. which is independent of χ. In the metric (3.2), we also assume invariance under two discrete transformations P1 and P2 deﬁned by P1(τ, χ, φ) = (−τ, −χ, −φ) , P2(τ, χ, φ) = (τ, χ + π, φ) . 3 Cohomogeneity-1 spacetime with rotating scalar ﬁeld (3.6) (3.6) The 1-forms (dτ, σ1, σ2, σ3) are transformed by P1 and P2 as The 1-forms (dτ, σ1, σ2, σ3) are transformed by P1 and P2 as P1(dτ, σ1, σ2, σ3) = (−dτ, −σ1, σ2, −σ3), P2(dτ, σ1, σ2, σ3) = (dτ, −σ1, −σ2, σ3) . (3.7) (3.7) Because of the invariance under P1 and P2, cross terms such as σ1σ2 do not appear in eq. (3.2). By examining boundary conditions, it turns out that the metric ansatz (3.2) is taken to be in a frame where asymptotic inﬁnity is rotating. We will search for black holes with a Killing horizon generated by ∂τ. This condition in turn enforces that f(rh) = g(rh) = 0. – 6 – For black hole solutions with α(r) ̸= 1 we must also satisfy h(rh) = 0 (see appendix A.4).3 Meanwhile, h(r) approaches a constant value Ωat inﬁnity r →∞, and the asymptotic form of the metric becomes For black hole solutions with α(r) ̸= 1 we must also satisfy h(rh) = 0 (see appendix A.4).3 Meanwhile, h(r) approaches a constant value Ωat inﬁnity r →∞, and the asymptotic form of the metric becomes ds2 ≃−r2dτ 2 + dr2 r2 + r2 4 n σ2 1 + σ2 2 + (σ3 + 2Ωdτ)2o , (3.8) (3.8) from which we see that the boundary metric is R(τ) × S3, but with rotation in the σ3 directions. This is the rotating frame at inﬁnity. The meaning of Ωbecomes clear by moving to the non-rotating frame at inﬁnity, which is the natural frame for interpreting conserved charges and other quantities of the black hole. We can switch to the non-rotating frame by applying the following coordinate transformation: JHEP05(2021)011 dt = dτ , dψ = dχ + 2Ωdτ . (3.9) (3.9) In the new frame, the horizon generator is written as4 ∂ ∂τ = ∂ ∂t + Ω ∂ ∂(ψ/2) . (3.10) (3.10) Therefore, Ωcorresponds to the angular velocity of the horizon. When Ω> 1, the norm of ∂τ, gττ, becomes positive at inﬁnity. This implies that there is no global time-like Killing vector in the domain of outer communications, and therefore the spacetime is non- stationary for Ω> 1. If α ̸= 1, the components of the new metric transformed from eq. (3.2) become explicitly time dependent. ( ) 4We use a canonically normalized angular coordinate ψ/2 ∈[0, 2π). 5See also ref. [34] for a ﬁve-dimensional cohomogeneity-1 geometry with periodic time dependence in asymptotically Poincaré AdS space with a S1 direction. 3If α(r) ̸= 1, the U(1)R isometry χ →χ + const. is broken, and therefore there is no continuous shift of r) that does not change the metric. ( ) g 4We use a canonically normalized angular coordinate ψ/2 ∈[0, 2π). 3 Cohomogeneity-1 spacetime with rotating scalar ﬁeld Thus our metric can be said to describe time periodic solutions, even though the metric ansatz is cohomogeneity-1.5 In our metric ansatz, we can therefore distinguish between time-periodic solutions (with α ̸= 1) and solutions that are stationary (with α = 1). Now we comment on the scalar ﬁeld (3.3). This ansatz is precisely the “double step- ping” ansatz (k decreases by 2 in the sum) introduced in the perturbative analysis of black resonators and geons [24]. Indeed, the modes in K decouple from those of its complement Kc = {j, j −1, · · · , −j} \ K. This fact ultimately stems from the discrete isometry P2 of the metric (3.2). Under P2, the Klein-Gordon equation for ⃗Π can be decomposed into even and odd parts. More speciﬁcally, because ⃗Dk ∝e−ikχ, the Wigner D-matrices with k ∈K and k ∈Kc acquire diﬀerent phase factors of ±1. The Klein-Gordon equations for k ∈K and k ∈Kc are therefore decoupled. In this paper, we will consider only k ∈K. Note also that we have deﬁned our ansatz with ﬁxed j and 2j + 1 multiplet. However, a solution with a particular j is also a solution to the theory with larger multiplets than 2j + 1, simply by setting the extra components of the multiplet to zero. Solutions with diﬀerent j can therefore be consistently compared with one another. – 7 – Finally, we show that the scalar ﬁeld ansatz (3.3) is consistent with the metric (3.2), i.e. the Einstein and Klein-Gordon equations reduce to a consistent set of ODEs. The Ein- stein equations from eq. (3.1) are given by Gµν −6gµν = Tµν, where the energy-momentum tensor is 1 Tµν = T(µν) −1 2gµνT , Tµν = ∂µ⃗Π∗· ∂ν⃗Π , T = gµνTµν . (3.11) (3.11) To derive the explicit expression of the energy-momentum tensor for eq. (3.3), it is convenient to introduce 1-forms σ± deﬁned by To derive the explicit expression of the energy-momentum tensor for eq. (3.3), it is convenient to introduce 1-forms σ± deﬁned by σ± = 1 2(σ1 ∓iσ2) = 1 2e∓iχ (∓idθ + sin θdφ) (3.12) (3.12) JHEP05(2021)011 d use the basis ea = {dτ, dr, σ+, σ−, σ3} (a = τ, r, +, −, 3). 3 Cohomogeneity-1 spacetime with rotating scalar ﬁeld Their dual vectors are given ea = {∂τ, ∂r, e+, e−, ∂χ} where and use the basis ea = {dτ, dr, σ+, σ−, σ3} (a = τ, r, +, −, 3). Their dual vectors are given by ea = {∂τ, ∂r, e+, e−, ∂χ} where e± = ¯ξ1 ± i¯ξ2 = ±R∓= e±iχ ±i∂θ + 1 sin θ∂φ −cot θ∂χ . (3.13) (3.13) One can check that ea µeµ b = δa b . One can check that ea µeµ b = δa b . (2.8) µ In this basis, the derivatives of the scalar ﬁeld can be evaluated by using eqs. (2.8) and (2.11) as ∂τ ⃗Π = 0 , ∂r⃗Π = X k∈K Φ′ k ⃗Dk , ∂+⃗Π = X k∈K ϵkΦk ⃗Dk−1 , ∂−⃗Π = − X k∈K ϵk+1Φk ⃗Dk+1 , ∂3⃗Π = −i X k∈K kΦk ⃗Dk , (3.14) (3.14) where ∂a ≡eµ a∂µ. where ∂a ≡eµ a∂µ. Some components of Tab vanish because of the double stepping coupling (3.4). For example, we ﬁnd Tr+ = X k,k′∈K ϵkΦ′ k′Φk ⃗D∗ k′ · ⃗Dk−1 = X k,k′∈K ϵkΦ′ k′Φkδk′,k−1 = 0 , (3.15) (3.15) where in the second equality we used eq. (2.14), and the last one follows from the fact that k′ and k −1 cannot be equal because of the double stepping of k (3.4). where in the second equality we used eq. (2.14), and the last one follows from the fact that k′ and k −1 cannot be equal because of the double stepping of k (3.4). To evaluate the non-vanishing components of Tab, we also use the orthogonality of the Wigner D-matrices (2.14). The upshot is that the energy momentum tensor is given by T(ab)eaeb = X k∈K Φ′ k 2dr2 −ϵk−1ϵkΦk−2Φk(σ2 + + σ2 −) + (ϵ2 k + ϵ2 k+1)Φ2 kσ+σ−+ k2Φ2 kσ2 3 . (3.16) (3.16) This result, invariant under SU(2)L, is consistent with the spacetime (3.2), and the Einstein and Klein-Gordon equations reduce to a consistent set of coupled ODEs. The explicit form of the equations of motion is summarized in appendix A, where technical details in solving the equations are also explained. – 8 – In our ansatz of the scalar ﬁeld (3.3), the conserved current of the complex scalar ﬁeld Jµ is given by In our ansatz of the scalar ﬁeld (3.3), the conserved current of the complex scalar ﬁeld Jµ is given by Jµdxµ ≡Im[ ⃗Π · ∂µ⃗Π∗]dxµ = X k∈K k2Φ2 kσ3 . (3.17) (3.17) We have Jψ ̸= 0 if and only if Φk ̸= 0 for k ̸= 0. This indicates that there is a rotating ﬂow of the scalar ﬁeld and it carries angular momentum when the scalar ﬁeld has non-trivial ψ-dependence. 4 Geons and superradiant instability of Myers-Perry-AdS Energy of geons Egeon as a function of the angular momentum J. 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 JHEP05(2021)011 Figure 1. Energy of geons Egeon as a function of the angular momentum J. Extreme MPAdS Gravitational perturbation Scalar ﬁeld perturbations 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 0 0.2 0.4 0.6 0.8 1 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.5 1 1.5 2 2.5 3 3.5 4 0.436 0.4365 0.437 0.4375 2 2.15 2.3 0.0914 0.0916 0.0918 0.05 0.055 0.06 Extreme MPAdS Gravitational perturbation Scalar field perturbation Figure 2. (Left) Onset of the superradiant instability of MPAdS against gravitational perturbation (red) and scalar ﬁeld perturbations with j = k = 9/2, 5 (blue) in the (Ω, rh)-plane. Above the red and blue curves, MPAdS is unstable to gravitational and scalar perturbations, respectively. The extreme limit of MPAdS is shown by the black curve. (Right) The same data as the left panel are shown in the (E −Egeon, J)-plane, though for the scalar ﬁeld perturbations only the onset of the mode with j = k = 9/2 is shown for visibility. 0 0.1 0.2 0.3 0.4 0.5 0.6 0 0.5 1 1.5 2 2.5 3 3.5 4 0.436 0.4365 0.437 0.4375 2 2.15 2.3 0.0914 0.0916 0.0918 0.05 0.055 0.06 Extreme MPAdS Gravitational perturbation Scalar field perturbation Extreme MPAdS Gravitational perturbation Scalar ﬁeld perturbations 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 0 0.2 0.4 0.6 0.8 1 Figure 2. (Left) Onset of the superradiant instability of MPAdS against gravitational perturbation (red) and scalar ﬁeld perturbations with j = k = 9/2, 5 (blue) in the (Ω, rh)-plane. Above the red and blue curves, MPAdS is unstable to gravitational and scalar perturbations, respectively. The extreme limit of MPAdS is shown by the black curve. (Right) The same data as the left panel are shown in the (E −Egeon, J)-plane, though for the scalar ﬁeld perturbations only the onset of the mode with j = k = 9/2 is shown for visibility. that breaks only the U(1)R isometry of the metric. Its onset mode of the gravitational per- turbation can be found in our ansatz by perturbing the metric function as α(r) = 1 + δα(r) about MPAdS and linearising the equation of motion (A.5) in δα(r). 4 Geons and superradiant instability of Myers-Perry-AdS JHEP05(2021)011 JHEP05(2021)011 If α(r) = 1 in eq. (3.2), there is an exact solution describing a rotating black hole with both angular momenta set equal. It is part of the Myers-Perry-AdS family of solutions, which we will abbreviate MPAdS. In our ansatz, the metric functions are g(r) = 1 −2µ(1 −a2) r2(1 + r2) + 2a2µ r4(1 + r2) , β(r) = 1 + 2a2µ r4 , h(r) = Ω− 2µa r4 + 2a2µ , f(r) = g(r) β(r) , α(r) = 1 . (4.1) (4.1) The event horizon r = rh is located at the largest root of g(rh) = 0, and the isometry group of this solution is R(τ) × SU(2)L × U(1)R. The solution is parametrised by µ and a, with µ = 0 corresponding to pure AdS. As written, Ωis merely a gauge parameter that allows us to move between rotating and non-rotating frames at inﬁnity. For consistency of notation and convenience, we set Ω= 2µa r4 h + 2a2µ (4.2) (4.2) so that Ωis the angular velocity of the horizon. For this choice, we have h(rh) = 0 and, thus, ∂τ becomes the horizon generator. MPAdS solutions are bounded by extremality, which occurs at q Ωextr = q 1 + 2r2 h √ 2rh . (4.3) (4.3) Before discussing perturbations of MPAdS, let us brieﬂy describe a family of solutions called geons. Geons are horizonless, nonlinear extensions of gravitational normal modes of pure AdS. Within our ansatz, these normal modes are given by a perturbation of the form α = 1 + δα(r) about AdS, with Ωappearing as an eigenvalue. Geons therefore carry angular momentum, with Ωas an angular frequency. Figure 1 is the energy of geons Egeon as a function of the angular momentum J. Later in this paper, we will primarily use the diﬀerence of the energy E from Egeon in ﬁgures for better visibility. Near vacuum AdS, the geons have frequency parameter Ω≃3/2, and hence the energy scales as Egeon ≃(3/2)J. Now let us return to perturbations of MPAdS black holes. When the MPAdS black hole has suﬃciently high angular frequency, it is unstable to superradiance against both gravitational and scalar ﬁeld perturbations. Among the unstable superradiant modes is one – 9 – 0 2 4 6 8 10 12 14 16 0 2 4 6 8 10 12 Figure 1. 4 Geons and superradiant instability of Myers-Perry-AdS The onset mode of the scalar ﬁeld perturbation is just given by the probe scalar ﬁeld satisfying the Klein-Gordon equation (A.5). In the MPAdS background, modes with diﬀerent k decouple in the Klein- Gordon equation. We will focus on modes with k = j, as these are the most dominant. In ﬁgure 2, we show results for gravitational and scalar ﬁeld onsets with j = k = 9/2 and j = k = 5. In the left ﬁgure, we show the onset of the instabilities in the (Ω, rh)- and (E −Egeon, J)-planes. The scalar and gravitational onset curves intersect at green dots. For reference, we also show where MPAdS is extremal, and where Ω= 1. Recall that MPAdS is unstable for Ω> 1, but not necessarily to the j = k = 9/2 and j = k = 5 modes. – 10 – Extreme MPAdS 0 1 2 3 4 5 6 0 0.1 0.2 0.3 0.4 0 1 2 3 4 5 6 7 8 9 Figure 3. Entropy of black resonators as a function of (E −Egeon, J). The upper solid curve indicates the onset of the gravitational superradiant instability of the MPAdS, where the black resonators branch oﬀ. The extreme MPAdS is shown by a black curve. In the upper-right region below the onset curve, we simply do not have numerical data. Extreme MPAdS 0 1 2 3 4 5 6 0 0.1 0.2 0.3 0.4 0 1 2 3 4 5 6 7 8 9 JHEP05(2021)011 Figure 3. Entropy of black resonators as a function of (E −Egeon, J). The upper solid curve indicates the onset of the gravitational superradiant instability of the MPAdS, where the black resonators branch oﬀ. The extreme MPAdS is shown by a black curve. In the upper-right region below the onset curve, we simply do not have numerical data. In the right ﬁgure, we show the same results in the (E −Egeon, J)-plane, but only show the result for the scalar for j = k = 9/2 for visibility. The onset of the instability for gravitational and scalar ﬁeld perturbations are shown by red and blue curves, respectively. These onset curves intersect at green dots. The extreme MPAdS is also shown by the black curve. Onset curves of scalar ﬁeld perturbations terminate at black dots on the extreme MPAdS. 4 Geons and superradiant instability of Myers-Perry-AdS In the insets in the right panel, we zoom in on the regions around the green and black dots for visibility. Note that, in the left inset, we take E −2.75J as the vertical axis for visibility. 7The normal mode frequency for the scalar ﬁeld with the quantum numbers (j, k) is given by Ω= (2 + j + n)/k where n is the radial overtone number. The lowest mode has n = 0. One can also see that k = j gives the lowest \|Ω\|. 5 Black resonators As mentioned in the previous section, MPAdS is unstable to superradiance against gravi- tational perturbations. A new family of cohomogeneity-1 solutions with α(r) ̸= 1 branches oﬀfrom the onset of the instability [23]. These onsets were given by the red curves la- belled “gravitational perturbations” in ﬁgure 2. Because α ̸= 1 for this new family, these black holes are time-periodic as seen in the non-rotating frame at inﬁnity and are known as black resonators. These black resonators have R × SU(2)L isometries. Like MPAdS black holes, these black resonators are a two-parameter family. The horizonless limit of black resonators are geons [12, 35–37], which we have already described in the previous section and in ﬁgure 1. Like black resonators, geons also have R × SU(2)L isometries and are time-periodic in the non-rotating frame at inﬁnity. In ﬁgure 3, the entropy of black resonators S is shown by the colour map as a function of (E −Egeon, J). The extreme MPAdS is shown by a black curve. Only MPAdS exists in the upper side of the black curve. The black resonators branch oﬀfrom the onset of the superradiant instability shown by the solid curve on the upper edge of the plotted region. In the upper-right white region below the onset curve, we do not have numerical data for black resonators. For a large J, the MPAdS at the onset is very close to the extremality, – 11 – and numerical construction of the black resonator becomes diﬃcult. In the limit of geons, E −Egeon →0, the entropy also approaches zero. In ref. [23], it has been found that the angular velocity of the black resonator and geon always satisﬁed Ω> 1. Therefore, the black resonator and geon are superradiant and non-stationary. 6 Hairy Myers-Perry-AdS and boson stars Thus far, we have discussed MPAdS, black resonators, and geons, which are all solutions that satisfy ⃗Π = 0. Now we turn to solutions with ⃗Π ̸= 0, beginning with those with α = 1. A special case of our ansatz (3.3) is given by the scalar ﬁeld with “single-k”: A special case of our ansatz (3.3) is given by the scalar ﬁeld with “singl JHEP05(2021)011 ⃗Π(τ, r, θ, φ, χ) = Φk(r) ⃗Dk(θ, φ, χ) (no summation) . (6.1) (6.1) In this case, the matter stress tensor eq. (3.16) reduces to In this case, the matter stress tensor eq. (3.16) reduces to T(ab)eaeb = Φ′ k 2dr2 + 1 4(ϵ2 k + ϵ2 k+1)Φ2 k(σ2 1 + σ2 2) + k2Φ2 kσ2 3 , (6.2) (6.2) where we returned to σ1,2 from σ±. In this expression, the coeﬃcients of σ1 and σ2 coincide, and therefore it has the invariance under U(1)R ∈SU(2)R generated by the angular momentum operator R3. The metric (3.2) therefore also has this isometry, which implies that α(r) = 1 in the single-k case. Superradiant instabilities can be induced by scalar ﬁelds as well as gravitational ﬁelds. These onset curves were shown earlier for the scalar ﬁeld with j = k = 9/2 and j = k = 5 by the blue curves in ﬁgure 2. For the scalar ﬁeld, we refer to the solutions branching oﬀfrom the onset of the superradiant instability as the hairy MPAdS black holes, as they contain scalar hair. Because black resonators can be interpreted as black holes with gravitational hair, hairy black holes are the scalar ﬁeld counterparts to black resonators. A hairy MPAdS black hole becomes a boson star in the horizonless limit. Boson stars can also be described as nonlinear scalar normal modes of pure AdS. Boson stars are therefore the scalar ﬁeld counterparts to geons. We obtain the perturbative solution of the boson star near pure AdS in appendix C, where we also discuss the large-j limit for the perturbative solution. In the following, we focus on the scalar ﬁeld with k = j, which is the most relevant mode for the superradiant instability for a given j. We note that the hairy MPAdS black hole with j = 1/2 was constructed in ref. [9].6 Our treatment (6.1) gives a generalization to j ≥1. 6The ansatz (6.1) for j = 1/2 was ﬁrst considered in ref. [38] for constructing rotating boson stars asymptotically ﬂat spacetime. 6 Hairy Myers-Perry-AdS and boson stars In ﬁgure 4(a), the energy of boson stars are shown as a function of the angular momen- tum J. The curves correspond to j = 1/2, 1, 3/2, · · · , 5 from left to right. The diﬀerence of the energy from that of the gravitational geon (see ﬁgure 1) is used in the vertical axis. These results near small E and J agree with perturbation theory about pure AdS. More iﬁ ll h l ﬁld (6 1) i h k j h h l l d Ω 1 2/j In ﬁgure 4(a), the energy of boson stars are shown as a function of the angular momen- tum J. The curves correspond to j = 1/2, 1, 3/2, · · · , 5 from left to right. The diﬀerence of the energy from that of the gravitational geon (see ﬁgure 1) is used in the vertical axis. tum J. The curves correspond to j = 1/2, 1, 3/2, · · · , 5 from left to right. The diﬀerence of the energy from that of the gravitational geon (see ﬁgure 1) is used in the vertical axis. These results near small E and J agree with perturbation theory about pure AdS. More speciﬁcally, the scalar ﬁeld (6.1) with k = j has the lowest normal mode at Ω= 1 + 2/j, from which the boson star branches oﬀ.7 For small J, the energy of the boson star is given These results near small E and J agree with perturbation theory about pure AdS. 6 Hairy Myers-Perry-AdS and boson stars 0 0.01 0.02 0.03 0 0.02 0.04 0.06 0.08 0.0326 0.033 0.0334 0.044 0.048 0.052 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0 2 4 6 8 10 12 0 1 2 3 4 5 6 7 8 0 0.10.20.30.40.5 0 0.5 1 1.5 2 2.5 1.3 1.34 1.38 1.42 Turning points of hairy MPAdS Onset of scalar ﬁeld instability of MPAdS Boson star (b) Entropy of hairy MPAdS (a) Energy of boson star b) Entropy of hairy MPAdS JHEP05(2021)011 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 6 7 8 Multi-valued Hairy MPAdS Extreme MPAdS Turning points of hairy MPAdS Onset of scalar field insability of MPAdS Boson star (c) Domain of hairy MPAdS 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 6 7 8 Multi-valued Hairy MPAdS Extreme MPAdS Turning points of hairy MPAdS Onset of scalar field insability of MPAdS Boson star (c) Domain of hairy MPAdS (c) Domain of hairy MPAdS Figure 4. (a) Energy of boson stars as a function of the angular momentum J. The existence multiple turning points are shown in the insets for j = 1/2. (b) Entropy of the hairy MPAdS Figure 4. (a) Energy of boson stars as a function of the angular momentum J. The existence of multiple turning points are shown in the insets for j = 1/2. (b) Entropy of the hairy MPAdS for k = j = 9/2, parametrized by J and E −Egeon. The solid curve on the left edge indicates the onset of the scalar ﬁeld superradiant instability of the MPAdS, from which the hairy MPAdS branches oﬀ. (c) Domain of existence of the hairy MPAdS. In orange and red regions, the hairy MPAdS exists. In the red region, the entropy and other physical quantities become multi-valued because of the turning points. by E ≃(1 + 2/j)J. Comparing this with that of the gravitational geon Egeon ≃(3/2)J, one ﬁnds that E −Egeon in small J is O(J2) for j = 4 and negative for j ≥9/2. As J is increased, there are turning points in the energy of boson stars, indicating the change of stability of the boson stars. It is common for solutions past the turning points to be unstable [39–42]. 6 Hairy Myers-Perry-AdS and boson stars More speciﬁcally, the scalar ﬁeld (6.1) with k = j has the lowest normal mode at Ω= 1 + 2/j, from which the boson star branches oﬀ.7 For small J, the energy of the boson star is given – 12 – 0 0.01 0.02 0.03 0 0.02 0.04 0.06 0.08 0.0326 0.033 0.0334 0.044 0.048 0.052 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0 2 4 6 8 10 12 (a) Energy of boson star 0 1 2 3 4 5 6 7 8 0 0.10.20.30.40.5 0 0.5 1 1.5 2 2.5 1.3 1.34 1.38 1.42 Turning points of hairy MPAdS Onset of scalar ﬁeld instability of MPAdS Boson star (b) Entropy of hairy MPAdS 0 0.1 0.2 0.3 0.4 0.5 0.6 0 1 2 3 4 5 6 7 8 Multi-valued Hairy MPAdS Extreme MPAdS Turning points of hairy MPAdS Onset of scalar field insability of MPAdS Boson star (c) Domain of hairy MPAdS Figure 4. (a) Energy of boson stars as a function of the angular momentum J. The existence of multiple turning points are shown in the insets for j = 1/2. (b) Entropy of the hairy MPAdS for k = j = 9/2, parametrized by J and E −Egeon. The solid curve on the left edge indicates the onset of the scalar ﬁeld superradiant instability of the MPAdS, from which the hairy MPAdS branches oﬀ. (c) Domain of existence of the hairy MPAdS. In orange and red regions, the hairy MPAdS exists. In the red region, the entropy and other physical quantities become multi-valued because of the turning points. 7 Hairy black resonator and graviboson star Finally, we consider the most general case in our ansatz (3.2) and (3.3) that has both α(r) ̸= 1 and ⃗Π(r) ̸= 0. Ref. [24] has located the onset of the superradiant instability of black resonators for scalar ﬁelds. At the onset of the instability, there is a τ-independent perturbation of the scalar ﬁeld in the form of eq. (3.3). This is expected to lead to a new family of black resonator solutions with a nontrivial scalar hair. Similarly, the hairy MPAdS black hole is expected to be unstable against gravitational perturbations with α(r) ̸= 1 (see appendix B), and a new family of black resonator solutions is expected to branch from the onset of this instability. We can therefore have scalar hair on black resonators, and gravitational “resonator”- type excitations on hairy MPAdS black holes. It turns out both of these excitations are part of the same family of solutions, which we call hairy black resonators. Hairy black resonators have their own family of horizonless solutions, which we refer to as graviboson stars, as they resemble a combination of a geon and a boson star. Figure 5(a) shows the domain of existence of the hairy black resonator. The hairy black resonator exists in the coloured “triangular” domain surrounded by the purple curves. The colour map corresponds to the angular velocity of the horizon. The top and bottom-left edges of the triangle correspond to the onset of instability of the hairy MPAdS and black resonator, respectively. The hairy MPAdS is unstable in the upper side of the top purple curve. The black resonator is unstable in the lower side of the bottom-left purple curve. The bottom-right edge is the horizonless limit of the hairy black resonator: graviboson star. We also show the equal-entropy-curve between the hairy MPAdS and black resonator by the orange curve. The angular velocity of the hairy black resonator always satisﬁes Ω> 1. Therefore, the hairy black resonator is also superradiant and non-stationary. We can now compare the entropy of the hairy black resonators to that of black res- onators and hairy black holes. Figure 5(b) gives a summary of the entropies of the black resonator (blue), hairy MPAdS (orange), and hairy black resonator (green) for j = 9/2, which is the smallest j for which black resonators can be unstable. Figure 5(c) corresponds to its slice at J = 0.8. 6 Hairy Myers-Perry-AdS and boson stars In the insets of the ﬁgure, we zoom in on the curve for j = 1/2 (here E −4.5J is used in the vertical axis for visibility). The curve is folded multiple times. The entropy of the hairy MPAdS for j = 9/2 is shown in ﬁgure 4(b). The angular velocity of the horizon is also shown by the colour map. For the result of j = 5, see section 9. The hairy MPAdS branches oﬀfrom the onset of the scalar ﬁeld superradiant instability denoted by the solid curve on the left edge of the plot region. Turning points of the energy also exist for hairy MPAdS as shown by the green curve, and as a result the entropy and other physical quantities of the hairy MPAdS become multi-valued. We – 13 – see Ω> 1, and this indicates that the hairy MPAdS and boson star are superradiant and non-stationary. Figure 4(c) shows the domain of existence of the hairy MPAdS in the (E −Egeon, J)- plane. In orange and red regions, the hairy MPAdS exists. In particular, in the red region, there are multiple solutions for a ﬁxed (E, J), and physical quantities are multi-valued. Our numerical calculation indicates that the curve of turning points terminates at the black dot: the intersecting point between the extreme MPAdS and the onset of scalar ﬁeld superradiant instability of MPAdS. A part of the turning points is shown by the dashed blue curve. We drew this part by interpolation of our numerical data. JHEP05(2021)011 7 Hairy black resonator and graviboson star We ﬁnd that the entropy of the hairy black resonator is never the largest among the available solutions. Instead, the most entropic solution is either a hairy MPAdS black hole or a black resonator (or MPAdS, but only in regions where none of the – 14 – 0 1 2 3 4 5 6 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 1.37 1.38 1.39 1.4 1.41 1.42 1.43 1.44 Boson star Hairy black resonator Onset of instability of black resonator Onset of instability of hairy MPAdS Onset of gravitational instability of MPAdS Graviboson star (a) Domain of hairy black resonator Black resonator Hairy MPAdS Hairy black resonator (b) Entropy Black resonator Hairy black resonator Hairy MPAdS (c) Cross section at J = 0.8 Figure 5. (a) Domain of existence of the hairy black resonator. It exists inside the “triangle” sur- rounded by purple curves. The equal-entropy-curve between the hairy MPAdS and black resonator is plotted by the orange curve. The angular velocity is shown by the color map. (b) Entropies of the black resonator (blue), hairy MPAdS (orange), and hairy black resonator (green) for j = 9/2. The entropy of the hairy black resonator is never the largest. (c) Cross section of (a) at J = 0.8. 0 1 2 3 4 5 6 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 1.37 1.38 1.39 1.4 1.41 1.42 1.43 1.44 Boson star Hairy black resonator Onset of instability of black resonator Onset of instability of hairy MPAdS Onset of gravitational instability of MPAdS Graviboson star Black resonator Hairy MPAdS Hairy black resonator Black resonator Hairy MPAdS Hairy black resonator (b) Entropy 0 1 2 3 4 5 6 -0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 1.37 1.38 1.39 1.4 1.41 1.42 1.43 1.44 Boson star Hairy black resonator Onset of instability of black resonator Onset of instability of hairy MPAdS Onset of gravitational instability of MPAdS Graviboson star JHEP05(2021)011 (a) Domain of hairy black resonator (a) Domain of hairy black resonator (b) Entropy (b) Entropy ( ) y ( ) py Black resonator Hairy black resonator Hairy MPAdS (c) Cross section at J = 0.8 Black resonator Hairy black resonator Hairy MPAdS (c) Cross section at J = 0.8 Hairy black resonator Figure 5. (a) Domain of existence of the hairy black resonator. It exists inside the “triangle” sur- rounded by purple curves. 7 Hairy black resonator and graviboson star 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 MPAdS Black resonator Hairy MPAdS (b) Black hole having maximal entropy 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 7 8 Turning points of hairy MPAdS Geon Boson star Extreme MPAdS Onset of instability of hairy MPAdS and black resonator Onset of gravitational and scalar field instability of MPAdS Graviboson star JHEP05(2021)011 (b) Black hole having maximal entropy (a) Phase diagram Figure 6. (a) Phase diagram of asymptotically AdS solutions in Einstein-multiplet complex scalar ﬁelds system for j = 9/2. (b) Black hole having maximal entropy among the MPAdS, hairy MPAdS, black resonator, and hairy black resonator for j = 9/2. One important diﬀerence in our solutions from those of [43] is the presence of a horizon. In order for solutions to remain steady-state (i.e. independent of τ), ﬁelds cannot pass through the horizon. This restricts the frequency of the ﬁelds to be multiple of the angular frequency of the horizon, and hence any multi-oscillating solutions must have commensurate frequencies. We expect non-commensurate multi-oscillating geons and boson stars to exist within this theory (3.1), but they would neither fall within our ansatz nor be the horizonless limit to a black hole. 7 Hairy black resonator and graviboson star The equal-entropy-curve between the hairy MPAdS and black resonator is plotted by the orange curve. The angular velocity is shown by the color map. (b) Entropies of the black resonator (blue), hairy MPAdS (orange), and hairy black resonator (green) for j = 9/2. The entropy of the hairy black resonator is never the largest. (c) Cross section of (a) at J = 0.8. Figure 5. (a) Domain of existence of the hairy black resonator. It exists inside the “triangle” sur- rounded by purple curves. The equal-entropy-curve between the hairy MPAdS and black resonator is plotted by the orange curve. The angular velocity is shown by the color map. (b) Entropies of the black resonator (blue), hairy MPAdS (orange), and hairy black resonator (green) for j = 9/2. The entropy of the hairy black resonator is never the largest. (c) Cross section of (a) at J = 0.8. other solutions exist). The entropy changes continuously, but the ﬁeld conﬁgurations are discontinuous across this transition. The hairy black resonator and graviboson star can be interpreted as a simple ex- ample of a multi-oscillating solution. Recall that the Wigner D-matrix depends on χ as Dk(θ, φ, χ) ∝e−ikχ. Then, in the non-rotating frame at inﬁnity (3.9), the scalar ﬁeld of the hairy black resonator can be written as ⃗Π(t, r, θ, φ, ψ) = X k∈K e2ikΩtΦk(r) ⃗Dk(θ, φ, ψ) . (7.1) (7.1) This has the eigenfrequencies ω = 2kΩ(k ∈K). Since the solution has periodic time dependence on several frequencies, it is multi-oscillating. In ref. [43], multi-oscillating boson stars with non-commensurate frequencies have been constructed by solving partial diﬀerential equations. In this paper, resonating solutions are obtained by solving ordinary diﬀerential equations although the frequencies are commensurate. – 15 – 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 7 8 Turning points of hairy MPAdS Geon Boson star Extreme MPAdS Onset of instability of hairy MPAdS and black resonator Onset of gravitational and scalar field instability of MPAdS Graviboson star (a) Phase diagram 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 MPAdS Black resonator Hairy MPAdS (b) Black hole having maximal entropy Figure 6. (a) Phase diagram of asymptotically AdS solutions in Einstein-multiplet complex scalar ﬁelds system for j = 9/2. (b) Black hole having maximal entropy among the MPAdS, hairy MPAdS, black resonator, and hairy black resonator for j = 9/2. 8 Phase diagram Finally, we put all the solutions together in a phase diagram in ﬁgure 6(a). We take j = 9/2 in an ensemble with ﬁxed-(E, J). We use E −Egeon as the vertical axis for visibility. Finally, we put all the solutions together in a phase diagram in ﬁgure 6(a). We take j = 9/2 in an ensemble with ﬁxed-(E, J). We use E −Egeon as the vertical axis for visibility. • The extreme MPAdS is located on the black curve. Regular MPAdS black holes exist above this curve, while the MPAdS develops a naked singularity below it. • The extreme MPAdS is located on the black curve. Regular MPAdS black holes exist above this curve, while the MPAdS develops a naked singularity below it. • The red curve and line are for the gravitational black resonators and geons. The curve on the top corresponds to the onset of the gravitational superradiant instability of the MPAdS. The black resonators branch oﬀfrom this curve to the bottom. The horizontal red line in the bottom (E = Egeon) expresses the family of the gravitational geons. The black resonators lie between the red curve and line. • The blue curves are associated with the hairy MPAdSs and boson stars. The upper- left part of the blue curve, from J = 0 to the black dot at J = 2.285, is the onset of the scalar ﬁeld superradiant instability on MPAdS black holes for k = j = 9/2. The onset coincides with the extreme MPAdS at the black dot, where the onset terminates. The family of hairy MPAdS black holes branches oﬀfrom this curve. – 16 – The other blue curve corresponds to the family of boson stars. There is a maximum in (E, J) at the top right, which is a turning point for the curve for the boson star. A collection of turning points for hairy MPAdSs, denoted by a blue dashed curve, extends from the top-right tip to the left as rh is increased. It appears to extend toward the black dot. The hairy MPAdSs exist in the region that is apparently enclosed by the blue curves for the onset and the boson star before the turning point, and the blue dashed curve. In the upper-right region enclosed by the upper boson star onset curve and the dashed curve, physical quantities of hairy MPAdSs become multi-valued. 8In the case of α(r) = 1, the equations of motion are identical for Φj(r) = cos λ φ(r), Φ−j(r) = sin λ φ(r) and Φk = 0 (\|k\| ̸= j) for any value of λ. The even and odd parity modes correspond to λ = ±π/2, and both modes give the same equations of motion. 8 Phase diagram JHEP05(2021)011 • The purple curves denote the boundary of the existing region for the hairy black res- onators and graviboson stars. The bottom-right side of the distorted purple triangle curve is the locations of the family of graviboson stars. The bottom-left side is the onset of the scalar ﬁeld superradiant instability of the black resonator [24]. The top side is the onset of the gravitational instability of hairy MPAdS. The hairy black resonators exist in the region enclosed by the purple curves. • The orange curve gives the location where the entropies of the black resonators and hairy MPAdSs become equal. Across the transition, the entropy is continuous, but the ﬁeld conﬁgurations are discontinuous. The hairy MPAdS has the higher entropy to the right of this curve, while the other side is dominated by the gravitational black resonators. Figure 6(b) is the phase diagram of the black hole solutions with the maximum entropy in the (E, J)-plane. The hairy black resonators never have the largest entropy and hence do not appear in this ﬁgure. Figure 6(b) is the phase diagram of the black hole solutions with the maximum entropy in the (E, J)-plane. The hairy black resonators never have the largest entropy and hence do not appear in this ﬁgure. 9 Phase diagram for higher j We can also obtain solutions for the multiplet complex scalar with j > 9/2. Here, we consider j = 5. Then, we need to consider 11-component complex scalar ﬁelds at least. Note that by setting one of the scalar multiplet components to zero, the j = 9/2 solution is also a solution for the theory with j = 5. So though we have deﬁned a scalar ﬁeld ansatz for particular j’s, the solutions with diﬀerent j can be consistently compared to one another, so long as we choose the scalar ﬁeld to have a suﬃciently large multiplet. In ref. [24], it was shown that, for an integer j, the scalar ﬁeld is decomposed into even and odd parity modes under the parity transformation P1 deﬁned in eq. (3.6). The even and odd parity modes satisfy Φ−k = Φk and Φ−k = −Φk (k ∈K), respectively. In this section, we only consider the even parity mode. In ﬁgure 7(a), we compare the entropies of the hairy MPAdS for j = 9/2 and j = 5.8 We ﬁnd that the solution for j = 5 has higher entropy than that for j = 9/2 at least in the region labeled as “hairy MPAdS” in ﬁgure 6(b). 8In the case of α(r) = 1, the equations of motion are identical for Φj(r) = cos λ φ(r), Φ−j(r) = sin λ φ(r) and Φk = 0 (\|k\| ̸= j) for any value of λ. The even and odd parity modes correspond to λ = ±π/2, and both modes give the same equations of motion. – 17 – This result suggests that, in a theory with a (2j′ + 1)-component complex scalar ﬁeld, a hairy MPAdS with j < j′ evolves into that with j = j′ by the superradiant instability in the region of a small angular momentum if we assume SU(2)L spacetime symmetry. (Note that the hairy MPAdS with j = j′ should be further unstable to SU(2)L-breaking perturbations [21].) Figure 7(b) is the phase diagram of solutions with j = 5. For the explanation of each curve, see section 8. The diagram is qualitatively similar to that for j = 9/2. Figure 7(c) is the phase diagram of the black hole solutions with the maximum entropy for j = 5. 9 Phase diagram for higher j The region in which the hairy MPAdS with j = 5 entropically dominates is bigger compared to the case of j = 9/2. This indicates that, the larger the quantum number j is, the wider the region covered by the hairy MPAdS will be in ﬁgure 6(b). If we extrapolate to arbitrarily large j, the black resonator would never dominate the phase diagram in a theory with an inﬁnite number of complex scalar ﬁelds. JHEP05(2021)011 10 Conclusion Curiously, though black resonators are unsta- ble to superradiant scalar perturbations and hairy MPAdS are unstable to superradiant – 18 – (a) Entropy for j = 9/2 and j = 5 -0.1 0 0.1 0.2 0.3 0.4 0.5 0 2 4 6 8 10 Turning points of hairy MPAdS Geon Boson star Onset of instability of hairy MPAdS and black resonator Onset of gravitational and scalar ﬁeld instability of MPAdS Graviboson star Extreme MPAdS (b) Phase diagram for j = 5 MPAdS Black resonator Hairy MPAdS -0.1 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 7 8 (c) Black hole having maximal entropy Figure 7. (a) Entropy of the hairy MPAdS for j = 9/2 (orange) and j = 5 (green). (b) Phase diagram of asymptotically AdS solutions in Einstein-multiple complex scalar ﬁelds system for j = 5. (c) Black hole having maximal entropy among the MPAdS, hairy MPAdS, black resonator, and hairy black resonator for j = 5. (a) Entropy for j = 9/2 and j = 5 -0.1 0 0.1 0.2 0.3 0.4 0.5 0 2 4 6 8 10 Turning points of hairy MPAdS Geon Boson star Onset of instability of hairy MPAdS and black resonator Onset of gravitational and scalar ﬁeld instability of MPAdS Graviboson star Extreme MPAdS (b) Phase diagram for j = 5 -0.1 0 0.1 0.2 0.3 0.4 0.5 0 2 4 6 8 10 Turning points of hairy MPAdS Geon Boson star Onset of instability of hairy MPAdS and black resonator Onset of gravitational and scalar ﬁeld instability of MPAdS Graviboson star Extreme MPAdS JHEP05(2021)011 b) Phase diagram for j = 5 MPAdS Black resonator Hairy MPAdS -0.1 0 0.1 0.2 0.3 0.4 0.5 0 1 2 3 4 5 6 7 8 (c) Black hole having maximal entropy (c) Black hole having maximal entropy (c) Black hole having maximal entropy (c) Black hole having maximal entropy Figure 7. (a) Entropy of the hairy MPAdS for j = 9/2 (orange) and j = 5 (green). (b) Phase diagram of asymptotically AdS solutions in Einstein-multiple complex scalar ﬁelds system for j = 5. (c) Black hole having maximal entropy among the MPAdS, hairy MPAdS, black resonator, and hairy black resonator for j = 5. gravitational perturbations, the solutions that branch from these instabilities (namely, the hairy black resonators) are never entropically dominant. 10 Conclusion To summarise our results, we have studied asymptotically global AdS solutions of Einstein gravity coupled to a (2j + 1) complex scalar multiplet within a cohomogeneity-1 ansatz. The following solutions are available within our ansatz: Myers-Perry-AdS black holes, black resonators (black holes with gravitational hair), black holes with scalar hair, and hairy black resonators (black holes with both gravitational and scalar hair). The latter three of these branch from various superradiant instabilities and have zero horizon limits that are geons, boson stars, and graviboson stars, respectively. The phase diagram of all of these solutions was shown in ﬁgures 6(a) and 7(b) for j = 9/2 and j = 5, respectively. The entropy of the hairy black resonator is never the largest among the three available solutions as shown in ﬁgure 6(b) for j = 9/2. This seems natural in the view of the perturbative stability. Inside the triangular region enclosed by purple curves in ﬁgure 6(a), both of the black resonator and hairy MPAdS is stable against corresponding perturbations. Thus, the black resonator and hairy MPAdS would not evolve into the hairy black resonator. This is consistent with the fact that the hairy black resonator is entropically subdominant. Finally, we were able to compare both j = 9/2 and j = 5 solutions, and we ﬁnd that the j = 5 hairy MPAdS solutions are dominant and cover a larger portion of phase space than those of j = 9/2. It is natural to expect that the trend continues to higher j. We can make this claim stronger by the following argument. The phase boundary between hairy MPAdS and black resonators always lies between two points: (1) The intersection between gravitational and scalar onsets of MPAdS, and (2) where the boson stars intersect geons. Results in ref. [24] suggest that the point (1) is located at a higher angular momentum for a higher j. Also, by explicitly constructing boson stars for j ≤11/2, we found that the same applies to the point (2) at least for j = 9/2, 5, 11/2. The study of time evolution of this system is an interesting future direction. If we assume SU(2)L-symmetry in the spacetime, a time-dependent ansatz for this system would give a 1 + 1 dimensional evolution system. 10 Conclusion Therefore, these unstable black resonators or hairy MPAdS cannot evolve to hairy black resonators. Instead, unstable black resonators will likely evolve towards hairy MPAdS, removing its gravitational hair. Similarly, unstable hairy MPAdS will evolve towards black resonators, shedding its scalar hair. Hairy black resonators themselves can (entropically) evolve to either black resonators or hairy MPAdS, most likely to whichever is dominant. In all of these case, scalars with k < j will be suppressed, and either the gravitational or k = j scalar ﬁeld instability will survive. It would be especially interesting to study the time evolution of a system with large j. As we have mentioned, a large j multiplet contains smaller j solutions within it, so the full system contains a tower of lower wavenumbers, many of which are unstable modes in black resonators or MPAdS. These modes have diﬀerent growth rates, with the largest – 19 – wavenumber typically being the slowest. However, the hairy black hole with the largest wavenumber is likely the most dominant entropically. A time evolution would therefore tell us how these competing instabilities interact with each other, and how a cascade to higher wavenumbers proceeds. Though the growth rates of high modes are extremely small and present a signiﬁcant numerical challenge, such a calculation seems more feasible in this 1+1 setting than the full 3+1 setting of Kerr-AdS. Furthermore, the high-wavenumbers are associated with angular directions rather than the radial direction. As the 1+1 equations do not directly see the angular gradients, the numerical resolution can be kept relatively low, allowing faster time evolution due to the larger Courant number. JHEP05(2021)011 A time-dependent ansatz for this system would give a 1 + 1 dimensional evolution system. In the discrete isometries (3.7), we can only assume P2-invariance for the time- dependent spacetime, and the equations of motion require a non-trivial cross term like γ(t, r)σ1σ2 in the metric for consistency. Despite the fact that we can construct solutions with several values of j, this comes at a cost. In particular, we would like to remind the reader that for each value of j we need 2j+1 complex scalar ﬁelds to make our co-homogeneity one ansatz work. Furthermore, the phases of each of these scalars must be ﬁne tuned so that the overall dependence in the angles do cancel. 10 Conclusion Perhaps more importantly, all these scalars are minimally coupled to gravity.9 One might ask whether such scalars are easy to come by in consistent reductions from some higher dimensional supergravity theory such as type IIB and the answer appears to be no. To our knowledge the largest known conjectured truncation of type IIB supergravity10 arises when considering compactiﬁcations of the form AdS5×S5, with the lower dimensional theory being ﬁve-dimensional N = 8 gauged supergravity comprising a total 42 scalars, 15 gauge ﬁelds and 12 form ﬁelds. However, these scalars appear to be non-minimally coupled to gravity, and to have very complicated potentials (see for instance [44]), thus giving very little hope that our model will ﬁnd a precise holographic realisation. 9One could potentially add a mass term, and much of our discussion would still go through. 10This has actually never been shown in full generality, partially because of the self dual condition imposed on the Ramond-Ramond F5 form ﬂux, even though interesting progress has been recently made in [44]. 10This has actually never been shown in full generality, partially because of the self dual condition impos on the Ramond-Ramond F5 form ﬂux, even though interesting progress has been recently made in [44]. 9One could potentially add a mass term, and much of our discussion would still go through. 10 A Technical details In this appendix, we collect technical details for solving the Einstein-complex scalar mul- tiplet system. Acknowledgments We would like to thank Oscar Dias for useful conversations. The work of T. I. was supported in part by JSPS KAKENHI Grant Number JP18H01214 and JP19K03871. The work of K. M. was supported in part by JSPS KAKENHI Grant Number JP18H01214 and JP20K03976. BW acknowledges support from ERC Advanced Grant GravBHs-692951 and MEC grant FPA2016-76005-C2-2-P. J. E. S. is supported in part by STFC grants PHY- 1504541 and ST/P000681/1. J. E. S. also acknowledges partial support from a J. Robert Oppenheimer Visiting Professorship. – 20 – A.1 Equations of motion (3.2) With our ansatz, the equations of motion are given by coupled ODEs. From eqs. (3.2) and (3.16), the trace T deﬁned in eq. (3.11) can be computed as T = X k∈K " (1 + r2)gΦ′ k 2 + 2 r2 α −1 α ϵk−1ϵkΦk−2Φk + 1 r2 α + 1 α (ϵ2 k + ϵ2 k+1)Φ2 k + 4 1 r2β − h2 (1 + r2)f ! k2Φ2 k # . A.1 Equations of motion (A.1) JHEP05(2021)011 For the metric ansatz (3.2), the Einstein equations Gµν −6gµν = Tµν become f′ = 1 r(1 + r2)2gα2(rβ′ + 6β)[4r2h2(α2 −1)2β + r(r2 + 1)g{r(1 + r2)fα′2β −r3h′2α2β2 −2(2 + 3r2)fα2β′} −4(1 + r2)f{6r2α2β(g −1) + 3gα2β + (α2 −αβ + 1)2 −4α2}] + 4rfβ rβ′ + 6β Trr , (A.2) ′ 1 [ 4 2h2( 2 1)2β ( ) g ( β β) + r(r2 + 1)g{r(1 + r2)fα′2β −r3h′2α2β2 −2(2 + 3r2)fα2β′} −4(1 + r2)f{6r2α2β(g −1) + 3gα2β + (α2 −αβ + 1)2 −4α2}] , (A.2) (A.2) g′ = 1 6r(1 + r2)2fα2β [−4r2h2(α2 −1)2β + r(1 + r2)g{−r(1 + r2)fα′2β + r3h′2α2β2 −(−r(1 + r2)f′ + 2f)α2β′} + 4(1 + r2)f{−6r2α2β(g −1) −3gα2β + α4 + 4α3β −5α2β2 −2α2 + 4αβ + 1}] , (A.3) + r(1 + r2)g{−r(1 + r2)fα′2β + r3h′2α2β2 −(−r(1 + r2)f′ + 2f)α2β′} + 4(1 + r2)f{−6r2α2β(g −1) −3gα2β + α4 + 4α3β −5α2β2 −2α2 + 4αβ + 1}] , (A.3) − 2 3r(1 + r2)2fβ [r2βTtt −4r2hβTt3 + 4(r2h2β −(1 + r2)f)T33] + 1 3r(1 + r2) α −1 α (T++ + T−−) −2 α + 1 α T+− h′′ = 1 2r2(1 + r2)α2βfg[8fh(α2 −1)2 (A ) h = 2r2(1 + r2)α2βfg[8fh(α 1) −r(1 + r2)h′α2{r(fg′β −f′gβ + 3fgβ′) + 10fgβ}] + 4(2hT33 −Tt3) r2(1 + r2)gβ , (A.4) 1 −r(1 + r2)h′α2{r(fg′β −f′gβ + 3fgβ′) + 10fgβ}] + 4(2hT33 −Tt3) r2(1 + r2)gβ , (A.4) α′′ = 1 2r2(1 + r2)2fαgβ [2r2(r2 + 1)2fgα′2β −r(r2 + 1)αα′{r(1 + r2)(fgβ)′ + 2(3 + 5r2)fgβ} −8(α2 −1){r2h2β(α2 + 1) −(1 + r2)fα(α −β) −(1 + r2)f}] − α r2(1 + r2)g α + 1 α (T++ + T−−) −2 α −1 α T+− , (A.5) α′′ = 1 2r2(1 + r2)2fαgβ [2r2(r2 + 1)2fgα′2β −r(r2 + 1)αα′{r(1 + r2)(fgβ)′ + 2(3 + 5r2)fgβ} −8(α2 −1){r2h2β(α2 + 1) −(1 + r2)fα(α −β) −(1 + r2)f}] − α r2(1 + r2)g α + 1 α (T++ + T−−) −2 α −1 α T+− , (A.5) (A.5) – 21 – – 21 – β′′ = 1 (2r2(1 + r2))fgα2β [−2r4gh′2α2β3 −rα2β′{r(1 + r2)(f′gβ + fg′β −fgβ′) + 2(3 + 5r2)fgβ} −8fβ(α4 + α3β −2α2β2 −2α2 + αβ + 1)] − 8 r2(1 + r2)T33 − β r2(1 + r2)g α −1 α (T++ + T−−) −2 α + 1 α T+− . A.1 Equations of motion (A.6) (A.6) For our scalar ﬁeld ansatz (3.3), the Klein-Gordon equation □⃗Π = 0 gives \|K\| = ⌊j + 1⌋ equations of the form JHEP05(2021)011 LkΦk + ck−1Φk−2 + ck+1Φk+2 = 0 , (A.7) (A.7) LkΦk + ck−1Φk−2 + ck+1Φk+2 = 0 , where Lk = (1 + r2)g d2 dr2 + " 1 + r2 2 f′ f + g′ g + β′ β + 3 + 5r2 r # g d dr −ϵ2 k + ϵ2 k+1 r2 α + 1 α −4k2 r2β + 4k2h2 (1 + r2)f , (A.8) (A.8) and ck = −ϵkϵk+1 r2 α −1 α . (A.9) (A.9) In (A.7), the mode coupling is “double-stepping” — the mode with k is coupled to those with k ± 2. We integrate these equations from the horizon r = rh (or the center r = 0 if the geometry is horizonless) to inﬁnity r →∞by using the 4th order Runge-Kutta method. The boundary conditions for solving the equations are given below. A.2 Boundary conditions at inﬁnity A.3 Boundary conditions at the origin for horizonless solutions JHEP05(2021)011 For horizonless solutions, we require regularity at the origin of AdS r = 0. To avoid a conical singularity at r = 0, we impose g, α, β →1 (r →0) . (A.14) (A.14) hen, from eq. (A.7), the regular solution of the scalar ﬁeld near r = 0 has the behavior Φk ∼r2j . (A.15) (A.15) To handle this behavior at arbitrary j, we ﬁnd it convenient to redeﬁne the scalar ﬁeld as o handle this behavior at arbitrary j, we ﬁnd it convenient to redeﬁne the scalar ﬁeld as Φk(r) = r2 1 + r2 !j Ψk(r) (A.16) (A.16) and solve the equations of motion for new variables X ≡(f, g, h, α, β, Ψk). They can be expanded near r = 0 as and solve the equations of motion for new variables X ≡(f, g, h, α, β, Ψk). They can be expanded near r = 0 as X(r) = ∞ X m=0 X2mr2m , (A.17) (A.17) where g0 = α0 = β0 = 1 as eq. (A.14). Substituting this expansion into eqs. (A.2)–(A.7) and specifying f0, h0, α2, β2, Ψk,0 as the input parameters, we can determine the higher order coeﬃcients X2m order by order. (In practice, we evaluated X2m for m ≤2.) Because of the rescaling (A.12), we can set f0 = 1 without loss of generality. To construct gravitational geons, we set the scalar ﬁeld zero, Ψk(r) = 0 for all k. Then, we have three free parameters to be speciﬁed at the origin, h0, α2, and β2, while there are two boundary conditions (A.13) at inﬁnity. Thus, the geons are obtained as a one-parameter family. We set α2 ̸= 0 as the input parameter and determine h0 and β2 by the shooting method so that eq. (A.13) is satisﬁed. To obtain a family of geons, we start from a normal mode of pure AdS where h0 = 3/2 and β2 = 0 and turn on a tiny α2. Once a solution is successfully obtained, we slightly vary the value of α2 as well as using the previous result as the initial guess for the next solution. We repeat this process and construct the geon solutions shown in ﬁgure 1. For boson stars, we set α(r) = 1 and Ψk(r) = 0 for k < j, which means that α2 = Ψk<j,0 = 0. A.2 Boundary conditions at inﬁnity We require the spacetime to be asymptotically AdS at inﬁnity. In the rotating frame at inﬁnity, the condition for the metric components is f, α, β →1, h →Ω (r →∞) , (A.10) (A.10) and then g →1 also follows from the other the equations of motion. The asymptotic value of h(r) actually corresponds to the angular velocity of the horizon Ωas explained in section 3. We also require that the massless scalar ﬁeld falls oﬀat inﬁnity, Φk →0 (r →∞) . (A.11) (A.11) In the interpretation of the gauge/gravity duality, this means that there is no external source for the dual scalar operators in the boundary ﬁeld theory. Thus, nontrivial scalar ﬁelds are spontaneously induced by the instability in the geometry. In the interpretation of the gauge/gravity duality, this means that there is no external source for the dual scalar operators in the boundary ﬁeld theory. Thus, nontrivial scalar ﬁelds are spontaneously induced by the instability in the geometry. However, imposing f →1 as r →∞is actually redundant because there is rescaling symmetry in eq. (3.2). By a coordinate transformation τ →c τ for a constant c, the line element is invariant if the metric components are rescaled as f(r) →f(r) c2 , h(r) →h(r) c . (A.12) (A.12) – 22 – – 22 – Hence, if we obtain a solution with f∞≡f(r = ∞) ̸= 1, we can rescale it so that the new f satisﬁes f →1 at inﬁnity. That is, we take c = √f∞in the above scaling equation. Therefore, when we solve the equations of motion, we only need to impose Hence, if we obtain a solution with f∞≡f(r = ∞) ̸= 1, we can rescale it so that the new f satisﬁes f →1 at inﬁnity. That is, we take c = √f∞in the above scaling equation. Therefore, when we solve the equations of motion, we only need to impose α, β →1 (r →∞) , (A.13) (A.13) and then we can apply the transformation (A.12) to obtain a rescaled solution satisfying f →1. A.3 Boundary conditions at the origin for horizonless solutions There are three free parameters h0, β2, Ψj,0 at the origin. At inﬁnity, we impose two conditions β →1 and Ψj →1. Similar to the case of geons, we set Ψj,0 ̸= 0 – 23 – as the input and start the shooting method from the initial guess given by the scalar ﬁeld normal mode in pure AdS with h0 = 1 + 2/j and β2 = 0. For the construction of graviboson stars, we have \|K\| + 3 free parameters at the origin h0, α2, β2, Ψk,0, and \|K\| + 2 conditions (A.11) and (A.13) at inﬁnity. We set Ψj,0 ̸= 0 as the input and determine the other \|K\| + 2 parameters by the shooting. The normal mode frequencies ω for scalar ﬁelds in gravitational geon backgrounds have been computed in ref. [24]. The onset for graviboson stars corresponds to ω = 0. We start from that point and turn on a tiny value of Ψj,0. 11The choice of F(r) can be arbitrary, but we assume F(rh) = 0 and F ′(rh) ̸= 0. A.4 Boundary conditions at the horizon JHEP05(2021)011 For black hole solutions, boundary conditions are imposed at the horizon r = rh. The ﬁeld variables Y ≡(f, g, h, α, β, Φk) can be expanded near the horizon as Y (r) = ∞ X m=0 Ym(r −rh)m , (A.18) (A.18) where f0 = g0 = 0. Substituting eq. (A.18) into the equation of motion for h(r) (A.4) and looking at the leading order, we obtain where f0 = g0 = 0. Substituting eq. (A.18) into the equation of motion for h(r) (A.4) and looking at the leading order, we obtain   (α0 −1)2 + 2α2 0 X k∈K k2Φ2 k,0   h0 = 0 . (A.19) (A.19) There are two possibilities for the solutions to this equation: (i) α0 = 1 and Φk,0 = 0, and (ii) h0 = 0. The case (i) is nothing but the MPAdS solution. Indeed, the horizon value h0 can be arbitrary because of the recovered U(1)R isometry. For hairy and resonating solutions, we consider (ii). Let us redeﬁne the ﬁeld variables as f(r) = F(r) ˜f(r) , g(r) = F(r)˜g(r) , h(r) = F(r)˜h(r) , (A.20) (A.20) where F(r) ≡1 −r6 h/r6. With the new variables, the near horizon expansion can be given by Z(r) = ∞ X m=0 Zm(r −rh)m , (A.21) (A.21) where Z = ( ˜f, ˜g, ˜h, α, β, Φk). This F(r) is chosen by hand so as to accommodate the near horizon behavior and employ ˜f0, ˜g0, ˜h0 ̸= 0.11 Substituting eq. (A.21) into eqs. (A.2)–(A.7) and taking ˜f0, ˜h0, α0, β0, Φk,0, rh as the input parameters, we can determine the higher order coeﬃcients Zm. (Practically in our calculations, we truncate the series to m ≤2.) Furthermore, we can set ˜f0 = 1 without loss of generality because of the rescaling symme- try (A.12). For black resonators, the scalar ﬁelds are trivial, Φk(r) = 0. There are four free parameters ˜h0, α0, β0, rh at the horizon and two conditions (A.13) at inﬁnity. Thus we need to specify two input parameters at the horizon, for which we choose α0 and rh, and then ˜h0 and β0 are determined by the shooting method. We start from the onset of the – 24 – superradiant instability of the MPAdS evaluated in [7, 24], where α0 = 1, and turn on a small deformation as α0 −1 ̸= 0. A.4 Boundary conditions at the horizon Once the shooting method converges and a black resonator solution is obtained, we slightly increase α0 −1 as well as using the previous data as the initial guess of the shooting method for the next solution. For hairy MPAdS, we set α(r) = 1 and Φk(r) = 0 for k < j. Therefore, we have α0 = 1 and Φk<j,0 = 0 at the horizon. We are left with four free parameters ˜h0, β0, Φj,0, rh at the horizon, and there are two conditions β →1 and Φj →0 at inﬁnity. We specify Φj,0 and rh as input parameters and determine ˜h0 and β0 by matching the boundary condition at inﬁnity. The scalar ﬁeld superradiant instability of the MPAdS [11, 24] is where the hairy solutions branch. JHEP05(2021)011 For hairy black resonators, there are \|K\| + 4 free parameters ˜h0, α0, β0, Φk,0, rh at the horizon and \|K\| + 2 conditions (A.11) and (A.13) at inﬁnity. There are two options in extending hairy black resonator solutions: from the scalar ﬁeld superradiant instability of black resonators, and from the gravitational one of hairy MPAdSs. We take the latter. The gravitational instability of the hairy MPAdS is studied in appendix B. We start the construction of the hairy black resonators from the onset of this instability. We specify α0 and rh as the input parameters and determine the others by the shooting method. Starting from the onset of the gravitational instability of the hairy MPAdS, we turn on a small α0 −1. We could also obtain the solutions branching oﬀfrom the scalar ﬁeld superradiant instability of black resonators evaluated in [24]. However, it turned out that the shooting method did not converge nicely around the onset of the instability, and therefore we resort to the other option. This problem is discussed in appendix A.6. A.5 Physical quantities Near the asymptotic inﬁnity r →∞, the asymptotic solutions of the metric components and scalar ﬁelds are f(r) = 1 + cf r4 + · · · , g(r) = 1 + cf + cβ r4 + · · · , h(r) = Ω+ ch r4 + · · · , α(r) = 1 + cα r4 + · · · , β(r) = 1 + cβ r4 + · · · , Φk(r) = ck r4 + · · · , (A.22) (A.22) where cf, ch, cα, cβ, and ck are the constants that are determined by matching the series with the bulk, and the source of Φk has already been set zero. As discussed in section 3, the asymptotic value of h(r) corresponds to the angular velocity Ω. The constant ck corresponds to the expectation value of the operator ⃗O dual to ⃗Π in the boundary theory as ⟨⃗O⟩= X k∈K ck ⃗Dk(θ, φ, χ) = X k∈K cke−2kΩt ⃗Dk(θ, φ, ψ) . (A.23) (A.23) This depends on both time and spatial coordinates on the boundary. Because the boundary source for the scalar ﬁeld is absent, the boundary energy- omentum tensor Tij can be given by [45–48]. 8πG5Tij = −r2 2 Ciρjσnρnσ r=∞ , (A.24) (A.24) – 25 – – 25 – where i and j run over the coordinates on the AdS boundary, nµ is the unit normal to a bulk r-constant surface, and Cµνρσ is the bulk Weyl tensor. Using eq. (A.22), we obtain where i and j run over the coordinates on the AdS boundary, nµ is the unit normal to a bulk r-constant surface, and Cµνρσ is the bulk Weyl tensor. Using eq. (A.22), we obtain 8πG5Tijdxidxj = 1 2(cβ −3cf)dτ 2 + 2chdτ(σ3 + 2Ωdτ) −cf + cβ 8 (σ2 1 + σ2 2) + cα 2 (σ2 1 −σ2 2) + 1 8(−cf + 3cβ)(σ3 + 2Ωdτ)2 . (A.25) (A.25) This is written in the rotating frame at inﬁnity (τ, χ). In the non-rotating frame (t, ψ), the boundary stress tensor is rewritten as JHEP05(2021)011 8πG5Tijdxidxj = 1 2(cβ −3cf)dt2 + 2chdt¯σ3 −cf + cβ 8 (¯σ2 1 + ¯σ2 2) + cα(e4iΩt¯σ2 + + e−4iΩt¯σ2 −) + 1 8(−cf + 3cβ)¯σ2 3 , (A.26) where ¯σi (i = 1, 2, 3) are the invariant one-forms deﬁned in the non-rotating frame: χ in (2.2) is replaced with ψ for ¯σi. A.5 Physical quantities The energy and angular momentum are given by E = Z dΩ3Ttt = π(cβ −3cf) 8G5 , J = − Z dΩ3Tt(ψ/2) = −πch 2G5 , (A.27) (A.27) where we deﬁne the angular momentum with respect to ψ/2 ∈[0, 2π). For black hole solutions, the entropy S and temperature T are given by where we deﬁne the angular momentum with respect to ψ/2 ∈[0, 2π). For black hole solutions, the entropy S and temperature T are given by where we deﬁne the angular momentum with respect to ψ/2 ∈[0, 2π). For black hole solutions, the entropy S and temperature T are given by S = π2r3 h p β(rh) 2G5 , T = (1 + r2 h) p f′(rh)g′(rh) 4π . (A.28) (A.28) For simplicity, we set G5 = 1 in this paper. We can easily recover the dependence on G5 by E →G5E, J →G5J, and S →G5S. A.6 Interpolation of hairy black resonator data When we construct the hairy black resonator, we take the route to extend the solution from the onset of instability of the hairy MPAdS. Near the onset, the shooting method converges well, and we were able to obtain hairy black resonators. As the solution approaches the onset of the instability of the black resonator, however, we ﬁnd that the shooting method fails to converge. Here we argue that the void, however, can be ﬁlled by interpolation. In ﬁgure 8, we show E of the hairy black resonator as a function of J for a ﬁxed horizon radius rh = 0.3. For visibility, we use E−1.412J as the vertical axis. The purple points denote the numerical data, and the green point is the onset of the scalar ﬁeld instability of the black resonator. Between these points, we were not able to obtain numerical solutions. There might be other solutions with similar parameters at the horizon. One of the candidates is the black resonator with the scalar hair with nontrivial radial overtones. Nevertheless, our data is ﬁtted well by a second order polynomial as shown in the black curve in the ﬁgure. We used the interpolated data in the corresponding region in ﬁgure 5(b). – 26 – 0.125 0.13 0.135 0.14 0.145 0.5 1 1.5 2 2.5 Interpolation by 2nd order polynomial Hairy black resonator Onset of instability of black resonator Figure 8. Energy of the hairy black resonator as a function of the angular momentum J for a ﬁxed horizon radius rh = 0.3. JHEP05(2021)011 Figure 8. Energy of the hairy black resonator as a function of the angular momentum J for a ﬁxed horizon radius rh = 0.3. B Instability of hairy MPAdS In this appendix, we consider the linear perturbation of the hairy MPAdS. To ﬁnd the onset of the instability, we only need to consider τ-independent perturbations and ﬁnd their normal modes. For the hairy MPAdS solutions, we have α(r) = 1 and Φk(r) = 0 (k < j), while the other functions have nontrivial r-dependence. Around this background, we perturb the variables as (f, g, h, α, β, Φk) →(f+δf, g+δg, h+δh, α+δα, β+δβ, Φk+δΦk) in eqs. (A.2)–(A.7) and keep the linear order in the perturbations. It turns out that the perturbation variables δα and δΦj−2 decouple from the others. Their perturbation equations are given by δα′′ = −1 2 ( (fgβ)′ fgβ + 2(3 + 5r2) r(1 + r2) ) δα′ − 8 (1 + r2)g ( β −2 r2β + 2h2 (1 + r2)f −T+− 4r2 ) δα −δT++ + δT−− r2(1 + r2)g , (B.1) δα = −2 ( fgβ + r(1 + r2) ) δα − 8 (1 + r2)g ( β −2 r2β + 2h2 (1 + r2)f −T+− 4r2 ) δα −δT++ + δT−− r2(1 + r2)g , (B.1) (B.1) j−2 = 4 p j(2j −1) r2 Φjδα , (B.2) (B.2) where T+−= −r2 4 " (1 + r2)gΦ′ j 2 + 4j2 1 r2β − h2 (1 + r2)f ! Φ2 j # , (B.3) δT±± = −2 q j(2j −1)ΦjδΦj−2 −r2 4 " (1 + r2)gΦ′ j 2 + 4j r2 Φ2 j + 4j2 1 r2β − h2 (1 + r2)f ! Φ2 j # δα . (B.4) (B.3) δT±± = −2 q j(2j −1)ΦjδΦj−2 −r2 4 " (1 + r2)gΦ′ j 2 + 4j r2 Φ2 j + 4j2 1 r2β − h2 (1 + r2)f ! Φ2 j # δα . (B.4) (B.4) We wish to ﬁnd normal mode solutions to these equations with the sourceless boundary condition at inﬁnity: δα →0 and δΦj−2 →0. On the black hole horizon, we require – 27 – regularity. Substituting δα = a0 + a1(r −rh) + · · · and δΦj−2 = φ0 + φ1(r −rh) + · · · into eqs. (B.1)–(B.2), we can obtain the regular series solution where ai and φi for i ≥1 are determined by a0 and φ0. We set a0 = 1 to ﬁx the scale of the linear perturbation. Then, φ0 is left as the free parameter on the horizon. B Instability of hairy MPAdS This parameter is tuned so that the boundary condition at inﬁnity, δΦj−2 →0, is satisﬁed. For a general hairy MPAdS background speciﬁed by the horizon value of the background scalar ﬁeld Φj\|r=rh, however, δα →0 is not realized at inﬁnity. The boundary condition for δα is satisﬁed only at special values of Φj\|r=rh. We monitor the value of δα\|r=∞by increasing Φj\|r=rh and search the value of Φj\|r=rh at which δα\|r=∞= 0 is satisﬁed. We repeat this process for each horizon radius rh. For j = 9/2, the result of the onset is the upper purple curve in ﬁgure 5(a). From ﬁgure 2(a), we know that MPAdS at the branching points of hairy MPAdS becomes unstable against gravitational perturbation for rh ≳0.37. This indicate that, in the upper side of the onset curve in ﬁgure 5(a), hairy MPAdS is unstable. JHEP05(2021)011 C Perturbative solution of boson star In this appendix, we perturbatively construct the boson stars for small deformations from pure AdS. This has been considered in similar contexts [12, 35, 36, 49–51]. In particular, we consider the large-j limit. C.1 Higher order perturbation The equations in the second order in ϵ are The equations in the second order in ϵ are The equations in the second order in ϵ are h′′ 2 + 5 rh′ 2 = 8j(j + 2) r2(1 + r2)φ2 , β′′ 2 + 3 + 5r2 r(1 + r2)β′ 2 − 8 r2(1 + r2)β2 = −4j(2j −1) r2(1 + r2) φ2 , g′′ 2 + 2(1 + 2r2) r(1 + r2) g2 + 1 3(1 + r2)β′ 2 + 10 3r(1 + r2)β2 = −8(j + 2r2) 3r(1 + r2)φ2 , f′ 2 + 2(1 + 2r2) r(1 + r2) g2 + 2 + 3r2 3(1 + r2)β′ 2 + 2 3r(1 + r2)β2 = −4(j −4r2) 3r(1 + r2)φ2 , (C.9) (C.9) where, in the right hand side, we have eliminated φ′ by using eq. (C.8). The solutions to them can be decomposed into particular and homogeneous solutions as where, in the right hand side, we have eliminated φ′ by using eq. (C.8). C.1 Higher order perturbation We consider the higher order perturbation around global AdS as Φj(r) = Φ1(r)ϵ + Φ3(r)ϵ3 + · · · (C.1) Φj(r) = Φ1(r)ϵ + Φ3(r)ϵ3 + · · · (C.1) (C.1) and       f(r) g(r) h(r) β(r)       =       1 1 Ω0 1       +       f2(r) g2(r) h2(r) β2(r)       ϵ2 + · · · . (C.2) (C.2) We set α(r) = 1 for the boson star. The fundamental normal mode frequency of the test scalar ﬁeld in pure AdS is Ω0 = 1 + 2/j. We set α(r) = 1 for the boson star. The fundamental normal mode frequency of the test scalar ﬁeld in pure AdS is Ω0 = 1 + 2/j. The equation in the ﬁrst order in ϵ is given by The equation in the ﬁrst order in ϵ is given by Φ′′ 1 + 3 + 5r2 r(1 + r2)Φ′ 1 −4{j(j + 1) −(4 + 3j)r2} r2(1 + r2)2 Φ1 = 0 . (C.3) (C.3) This can be solved by Φ1(r) = φ(r) ≡ 1 (1 + r2)2 r2 1 + r2 !j , (C.4) (C.4) which is regular at the origin (r = 0) and inﬁnity (r = ∞): which is regular at the origin (r = 0) and inﬁnity (r = ∞): φ(r) ∼r2j (r →0) , φ(r) ∼1 r4 (r →∞) . (C.5) (C.5) – 28 – – 28 – The other solution to eq. (C.3) is he other solution to eq. (C.3) is ˜φ(r) = φ(r) Z r r3(1 + r2)φ2 . (C.6) (C.6) However, this is singular at the origin and inﬁnity as ˜φ(r) ∼r−2j−2 (r →0) , ˜φ(r) ∼1 (r →∞) . (C.7) (C.7) Therefore, we adopt φ (C.4) as the ﬁrst order solution. The solution satisﬁes JHEP05(2021)011 JHEP05(2021)011 φ′ = 2(j −2r2) r(1 + r2) φ . (C.8) (C.8) We will use this equation to eliminate φ′ in the following calculations. We will use this equation to eliminate φ′ in the following calculations. We will use this equation to eliminate φ′ in the following calculations. The equations in the second order in ϵ are We will use this equation to eliminate φ in the following calculations. C.1 Higher order perturbation The solutions to them can be decomposed into particular and homogeneous solutions as       f2 g2 h2 β2       =       fp 2 gp 2 hp 2 βp 2       +       fhom 2 ghom 2 hhom 2 βhom 2       , (C.10) where       f2 g2 h2 β2       =       fp 2 gp 2 hp 2 βp 2       +       fhom 2 ghom 2 hhom 2 βhom 2       , (C.10) (C.10) where hp 2 = 2j(j + 1) "Z r 0 φ2 r(1 + r2)dr −1 r4 Z r 0 r3φ2 1 + r2 dr # , βp 2 = j(2j −1) " 1 r4 Z r 0 rF(r)φ2dr −F(r) Z r 0 φ2 r3 dr # , gp 2 = − 1 3(1 + r2) βp 2 + 8 r2 Z r 0 {(j + 2r2)rφ2 + rβp 2}dr , fp 2 = −2 + 3r2 3(1 + r2)βp 2 −2 Z r 0 1 r(1 + r2) (1 + 2r2)gp 2 + 2 3(j −4r2)φ2 + 1 3(1 + r2)βp 2 dr , (C.11) (C.11) – 29 – and and hhom 2 = Ch , βhom 2 = CβA(r) , ghom 2 = Cβ (4r2 + 3)A(r) −4r2 3(1 + r2) , fhom 2 = Cf + Cβ r2A(r) 3(1 + r2) . (C.12) hhom 2 = Ch , βhom 2 = CβA(r) , (C ) (C.12) Here, we deﬁned A(r) ≡1 −2 r2 + 2 ln(1 + r2) r4 . (C.13) (C.13) Since we set the lower bound of the integral at r = 0, the regularity at the origin is guaranteed in these expressions. Integration constants Cβ and Cf will be chosen so that β2 →0 and f2 →0 (r →∞) are satisﬁed. Ch will be determined from the third order equation. C.1 Higher order perturbation JHEP05(2021)011 The third order equation is Φ′′ 3 + 3 + 5r2 r(1 + r2)Φ′ 3 −4{j(j + 1) −(4 + 3j)r2} r2(1 + r2)2 Φ3 = S3 , (C.14) (C.14) where S3 = 4φ (1 + r2)2 " (j + 2)2f2 −4r4 −2r2 −5jr2 + j2 r2 g2 −2j(j + 2)h2 −j2(1 + r2) + 2r2 −j 2 β2 + j(j −2r2) 2 φ2 # . (C.15) S3 = 4φ (1 + r2)2 " (j + 2)2f2 −4r4 −2r2 −5jr2 + j2 r2 g2 −2j(j + 2)h2 −j2(1 + r2) + 2r2 −j r2 β2 + j(j −2r2) r2 φ2 # . (C.15) We have eliminated f′ 2, g′ 2 and φ′ in the source term by using eqs. (C.8) and (C.9). The solution in this order is written as Φ3 = Cφφ(r) −φ(r) Z r 0 r3(1 + r2)S3 ˜φ(r)dr + ˜φ(r) Z r 0 r3(1 + r2)S3φ(r)dr . (C.16) Φ3 = Cφφ(r) −φ(r) Z r 0 r3(1 + r2)S3 ˜φ(r)dr + ˜φ(r) Z r 0 r3(1 + r2)S3φ(r)dr . (C.16) (C.16) The ﬁrst and second terms are regular at the origin and inﬁnity. The last term is also regular at the origin but it behaves as ∼1 at the inﬁnity. To satisfy the source free boundary condition at the inﬁnity, therefore, we need The ﬁrst and second terms are regular at the origin and inﬁnity. The last term is also regular at the origin but it behaves as ∼1 at the inﬁnity. To satisfy the source free boundary condition at the inﬁnity, therefore, we need Z ∞ 0 r3(1 + r2)S3φ(r)dr = 0 . (C.17) (C.17) This equation determines Ch as announced. This equation determines Ch as announced. This equation determines Ch as announced. This equation determines Ch as announced. C.2 Large-j expansion of the perturbative solution C.2 Large-j expansion of the perturbative solution C.2 Large-j expansion of the perturbative solution The integral expressions of the perturbative results obtained above can be evaluated in the large-j limit in powers of 1/j. The integral expressions of the perturbative results obtained above can be evaluated in the large-j limit in powers of 1/j. For the large-j expansion, it is convenient to introduce a new coordinate x as r2 1 + r2 = exp(−x2/j) . (C.18) (C.18) In the new coordinate, r = 0 and ∞correspond to x = ∞and 0, respectively. In terms of x, the ﬁrst order solution (C.4) is rewritten as he ﬁrst order solution (C.4) is rewritten as φ = (1 −e−x2/j)2e−x2 . (C.19) (C.19) – 30 – – 30 – Because of the exponential factor e−x2, this function is highly suppressed in x ≫1, i.e. r ∼0. Assuming x ≲1, we can expand the prefactor (1 −e−x2/j)2 by 1/j as Because of the exponential factor e−x2, this function is highly suppressed in x ≫1, i.e. r ∼0. Assuming x ≲1, we can expand the prefactor (1 −e−x2/j)2 by 1/j as φ = e−x2 x4 j2 −x6 j3 + 7x8 12j4 −x10 4j5 + · · · ! . (C.20) (C.20) Let us consider the large-j approximation of the second order solution. For example, the ﬁrst term in hp 2 (C.11) is written as Let us consider the large-j approximation of the second order solution. For example, the ﬁrst term in hp 2 (C.11) is written as JHEP05(2021)011 JHEP05(2021)011 Z r 0 φ2 r(1 + r2)dr = 1 j Z ∞ x x(1 −e−x2/j)4e−2x2dx (C.21) (C.21) The integrand contains an exponential factor e−2x2. Hence, we assume x ≲1 and expand the integrand by 1/j as The integrand contains an exponential factor e−2x2. Hence, we assume x ≲1 and expand the integrand by 1/j as Z ∞ x e−2x2 x9 j5 −2x11 j6 + 13x13 6j7 −5x15 3j8 + · · · ! dx = e−2x2 1 8j5 (2x8 + 4x6 + 6x4 + 6x2 + 3) Z ∞ x e−2x2 x9 j5 −2x11 j6 + 13x13 6j7 −5x15 3j8 + · · · ! C.2 Large-j expansion of the perturbative solution As a result, we have e2x2hp 2 = 1 4j3 (x2 + 1)(2x4 + 3x2 + 3) −1 8j4 (6x8 + 16x6 + 31x4 + 36x2 + 18) + 1 48j5 (30x10 + 89x8 + 216x6 + 393x4 + 450x2 + 225) − 1 96j6 (36x12 + 120x10 + 343x8 + 804x6 + 1425x4 + 1620x2 + 810) + 1 2880j7 (516x14 + 1930x12 + 6336x10 + 17805x8 (C.23) (C.23) + 1 2880j7 (516x14 + 1930x12 + 6336x10 + 17805x8 + 41040x6 + 71775x4 + 81270x2 + 40635) + · · · , – 31 – e2x2βp 2 = −1 4j3 x2 + 1 2x4 + 3x2 + 3 + 1 8j4 (6x8 + 24x6 + 47x4 + 48x2 + 18) − 1 48j5 (30x10 + 161x8 + 456x6 + 3(259 + 24y)x4 + 666x2 + 225) + 1 96j6 (36x12 + 240x10 + 887x8 + 12(181 −12y)x6 + 3(1151 + 144y)x4 + 2520x2 + 810) − 1 2880j7 (516x14 + 4090x12 + 18576x10 (C.24) e2x2βp 2 = −1 4j3 x2 + 1 2x4 + 3x2 + 3 + 1 8j4 (6x8 + 24x6 + 47x4 + 48x2 + 18) − 1 5 (30x10 + 161x8 + 456x6 + 3(259 + 24y)x4 + 666x2 + 225) − 1 48j5 (30x10 + 161x8 + 456x6 + 3(259 + 24y)x4 + 666x2 + 225) + 1 96j6 (36x12 + 240x10 + 887x8 + 12(181 −12y)x6 (C.24) (C.24) + 3(1151 + 144y)x4 + 2520x2 + 810) JHEP05(2021)011 JHEP05(2021)011 + 105(565 + 24y)x8 + 540(245 −24y)x6 + 675(299 + 40y)x4 + 129870x2 + 40635) + · · · , e2x2gp 2 = −1 4j4 x2(x4 −1)(2x2 + 5) + 1 8j5 x2(8x8 + 29x6 + 5x4 −(37 + 16y)x2 −30) − 1 48j6 x2(52x10 + 243x8 + 169x6 (C.25) + 1 8j5 x2(8x8 + 29x6 + 5x4 −(37 + 16y)x2 −30) − 1 48j6 x2(52x10 + 243x8 + 169x6 −(241 −72y)x4 −96(8 + 3y)x2 −375) + 1 92j7 x2(80x12 + 454x10 + 531x8 −8(14 −11y)x6 −(1853 −432y)x4 −5(743 + 240y)x2 −1350) + · · · , (C.25) (C.25) j −(241 −72y)x4 −96(8 + 3y)x2 −375) + 1 92j7 x2(80x12 + 454x10 + 531x8 −8(14 −11y)x6 −(1853 −432y)x4 −5(743 + 240y)x2 −1350) + · · · , e2x2fp 2 = 1 4j3 (x2 + 1)(2x4 + 3x2 + 3) −1 8j4 (10x8 + 18x6 + 27x4 + 30x2 + 18) + 1 48j5 (78x10 + 95x8 + 150x6 + 3(81 −8y)x4 + 342x2 + 225) − 1 96j6 (140x12 + 102x10 + 105x8 (C.26) (C.26) 96j + 186x6 + 3(191 −48y)x4 + 1170x2 + 810) + 1 2880j7 (2916x14 + 910x12 −2214x10 −15(549 + 8y)x8 −7530x6 + 75(221 −120y)x4 + 56970x2 + 40635) + · · · , where we deﬁned where we deﬁned y = e2x2Ei1(2x2) , Ei1(z) = Z ∞ 1 e−tz t dt . C.2 Large-j expansion of the perturbative solution dx = e−2x2 1 8j5 (2x8 + 4x6 + 6x4 + 6x2 + 3) −1 8j6 (4x10 + 10x8 + 20x6 + 30x4 + 30x2 + 15) + 13 96j7 (4x12 + 12x10 + 30x8 + 60x6 + 90x4 + 90x2 + 45) − 5 96j8 (8x14 + 28x12 + 84x10 + 210x8 + 420x6 + 630x4 + 630x2 + 315) + · · · . (C.22) (C.22) We can do the same procedure for the other integrals in eq. (C.11). Since the integrals always produce the exponential factor e−2x2, the functions of r outside of the integrals can be expanded by 1/j after changing coordinates from r to x. C.2 Large-j expansion of the perturbative solution (C.27) (C.27) At the origin and inﬁnity, this behaves as At the origin and inﬁnity, this behaves as At the origin and inﬁnity, this behaves as y ≃−γ −ln 2 −2 ln x (x →0) , y ≃ 1 2x2 (x →∞) , (C.28) (C.28) – 32 – – 32 – where γ is Euler’s constant. Near the inﬁnity (x = 0), the second order solutions approach where γ is Euler’s constant. Near the inﬁnity (x = 0), the second order solutions approach hp 2 → 3 4j3 −9 4j4 + 75 16j5 −135 16j6 + 903 64j7 + · · · , (C.29) βp 2 →−3 4j3 + 9 4j4 −75 16j5 + 135 16j6 −903 64j7 + · · · , (C.30) hp 2 → 3 4j3 −9 4j4 + 75 16j5 −135 16j6 + 903 64j7 + · · · , (C.29) βp 2 →−3 4j3 + 9 4j4 −75 16j5 + 135 16j6 −903 64j7 + · · · , (C.30) gp 2 →0 , (C.31) fp 2 → 3 4j3 −9 4j4 + 75 16j5 −135 16j6 + 903 64j7 + · · · . (C.32) (C.29) g2 →0 , (C 3 ) fp 2 → 3 4j3 −9 4j4 + 75 16j5 −135 16j6 + 903 64j7 + · · · . (C.32) Therefore, from the boundary condition for the second order perturbation, β2 →0 and f2 →0 at r →∞, we can determine the integration constants Cβ and Cf as JHEP05(2021)011 JHEP05(2021)011 Cβ = 3 4j3 −9 4j4 + 75 16j5 −135 16j6 + 903 64j7 + · · · , Cf = −4 3 3 4j3 −9 4j4 + 75 16j5 −135 16j6 + 903 64j7 + · · · . (C.33) (C.33) To determine Ch, we substitute the particular solutions (C.23)–(C.26) and homogeneous solutions (C.12) into eq. (C.17). Then, expanding the integrand by 1/j after moving to the x-coordinate, we can carry out the integration. As the result, we obtain Ch as To determine Ch, we substitute the particular solutions (C.23)–(C.26) and homogeneous solutions (C.12) into eq. (C.17). Then, expanding the integrand by 1/j after moving to the x-coordinate, we can carry out the integration. C.2 Large-j expansion of the perturbative solution As the result, we obtain Ch as Ch = −3 4j3 + 9 4j4 −3 2j5 29 16 + ln(eγj) + 21 4j6 −137 448 + ln(eγj) −12 j7 −16079 12288 + ln(eγj) + · · · . (C.34) −12 j7 −16079 12288 + ln(eγj) + · · · . (C.34) Near the inﬁnity, the second order solutions are expanded as Near the inﬁnity, the second order solutions are expanded as f2 = cf r4 + · · · , g2 = cg r4 + · · · , h2 = Ω2 + ch r4 + · · · , β = cβ r4 + · · · , (C.35) (C.35) where cf = −1 4j −3 8j2 + 1 2j3 9 8 + ln(2eγj) −3 2j4 −13 48 + ln(2eγj) + 25 8j5 −623 600 + ln(2eγj) + · · · , cg = −2 j2 + 2 j3 (1 + ln(2eγj)) −6 j4 −1 2 + ln(2eγj) + 25 2j5 −77 60 + ln(2eγj) + · · · , ch = −1 4j + 5 8j2 −19 16j3 + 65 32j4 −211 64j5 + · · · , cβ = 1 4j −13 8j2 + 3 2j3 23 24 + ln(2eγj) −9 2j4 −83 144 + ln(2eγj) + 75 8j5 −273 200 + ln(2eγj) + · · · . (C.36) j j −6 j4 −1 2 + ln(2eγj) + 25 2j5 −77 60 + ln(2eγj) + · · · , (C.36) (C.36) ch = −1 4j + 5 8j2 −19 16j3 + 65 32j4 −211 64j5 + · · · , j j j −9 2j4 −83 144 + ln(2eγj) + 75 8j5 −273 200 + ln(2eγj) + · · · . – 33 – The second order contribution of the normal mode frequency is The second order contribution of the normal mode frequency is Ω2 = −3 2j5 −21 16 + ln(eγj) + 21 4j6 −857 448 + ln(eγj) −12 j7 −30527 12288 + ln(eγj) + · · · . C.2 Large-j expansion of the perturbative solution (C.37) (C.37) From cf, cβ, ch, we can compute the second order contribution of the mass E2 and angular momentum J2 of the boson star as 8G π E2 = cβ −3cf = 1 j −1 2j2 −1 4j3 + 11 8j4 −49 16j5 + · · · , (C.38) 8G π J2 = −4ch = 1 j −5 2j2 + 19 4j3 −65 8j4 + 211 16j5 + · · · . (C.39) (C.38) JHEP05(2021)011 (C.39) One can check that these satisfy the ﬁrst law at all orders in the 1/j-expansion: E2 = Ω0J2 . (C.40) (C.40) Open Access. 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https://openalex.org/W3012898102	http://www.odermatol.com/odermatology/20202/16.Syringocystadenoma-ElFaqyrI.pdf	English	null	Syringocystadenoma papilliferum presented as an ulcerated nodule of the vulva in a patient with Neurofibromatosis type 1	Nasza Dermatologia Online	2,020	cc-by	1,872	How to cite this article: El Faqyr I, Dref M, Zahid S, Oualla J, Mansouri N, Rais H, Hocar O, Amal S. Syringocystadenoma papilliferum presented as an ulcerated nodule of the vulva in a patient with Neuroﬁ bromatosis type 1. Our Dermatol Online. 2020;11(2):174-176. Submission: 19.12.2019; Acceptance: 14.02.2020 DOI: 10.7241/ourd.20202.16 INTRODUCTION hyperpigmented macules with well-defined borders over the body, corresponding to café-au-lait macules (Fig. 1). Skinfold freckling were present all over the trunk (Fig. 1). Examination of the genital area objectified a pinkish to erythematous bordered elevated ulcerated nodule measuring about 1 cm in diameter, unpainful, located in the inside of the labia majora (Fig. 2). There were no other genital or perineal lesions. Ophthalmological examination found several bilateral Lisch nodules. The patient meted National Institutes of Health criteria for NF14 (4/7 criteria). Other physical examination showed dosrsolumbar scoliosis. The vulvar lesion was fully excised, histopathology revealed a tumor with endophytic configuration, composed of papillary projections and ductal-like structures that extend as invaginations from the surface epithelium into the underlying dermis. The papillary projections covered by regular cubic epithelium. The ducts and papillary projections are covered by an inner layer of columnar epithelium and an outer layer of cuboidal or flattened Syringocystadenoma papilliferum (SCAP) is an uncommon benign adnexal tumor, which frequently arises from a nevus sebaceous. This tumor occurs usually at birth or during early childhood. It’s most commonly located on the scalp or face, however SCAP in other areas have been reported [1]. Location in the vulva is rare and had only been reported in very few cases[2]. Neurofibromatosis type1 (NF1) is a frequent neurocutaneous syndrome that predisposes for various benign and malignant tumors. We report a case of SCAP located in the vulva in a NF1 patient. ABSTRACT A 36-year-old female presented to the dermatology service with a pinkish to erythematous ulcerated nodule measuring about 1 cm in diameter, located on the labia majora. The lesion has been slowly increasing in size. Physical examination found features compatible with the diagnosis of neurofibromatosis type 1. Excisional biopsy demonstrated the histological picture of syringocystadenoma papilliferum. The interest of this report lies in the rarity of syringocystadenoma papilliferum, its unusual presentation in the vulva, and it’s association with neurofibromatosis type 1. To our knowledge, this association has not been described in the medical literature. Key words: Syringocystadenoma papilliferum; Neurofibromatosis type 1; Vulva Our Dermatology Online Our Dermatology Online Case Report Iyda El Faqyr1, Maria Dref2, Sara Zahid1, Jamila Oualla1, Nabil Mansouri2, Hanane Rais2, Ouafa Hocar1, Said Amal1 1Department of Dermatology, Mohammed VI University Hospital, University Cadi Ayyad, Marrakech, Morocco, 2Department of Pathology, Mohammed VI University Hospital, University Cadi Ayyad, Marrakech, Morocco Corresponding author: Dr. Iyda El Faqyr, E-mail: iydaelfaqyr@gmail.com Corresponding author: Dr. Iyda El Faqyr, E-mail: iydaelfaqyr@gmail.com Syringocystadenoma papilliferum presented as Syringocystadenoma papilliferum presented as an ulcerated nodule of the vulva in a patient with an ulcerated nodule of the vulva in a patient with Neurofi bromatosis type 1 Neurofi bromatosis type 1 Iyda El Faqyr1, Maria Dref2, Sara Zahid1, Jamila Oualla1, Nabil Mansouri2, Hanane Rais2, Ouafa Hocar1, Said Amal1 DISCUSSION Syringocystadenoma papilliferum is presumably derived from pluripotent cells and differentiates toward eccrine and/or apocrine apparatuses [3]. There is also a possibility that SCAP can resemble lesions of the breasts deriving from the supernumerary mammary tissues from milk ridges. In some individuals those ridges may not regress completely during embryological development forming breast-like tissue [2]. The most frequent location is the head and neck. Less frequently involved sites are face, chest, abdomen, arm, thighs, and perineum, location on vulva had only reported in few cases. The clinical presentation varies widely, making the clinical diagnosis of SCAP quite difficult. The tumor can present as a solitary nodule or greyish-brown papule with smooth and flat dome. Large nodules can ulcerate. Main dermoscopic features are exophytic papillary structures and polymorphous vessels1. Histopathologic appearance is uniform and characteristic the basis of diagnosis [3]. It typically shows varying degrees of papillomatosis along with cystic invaginations and malformed sebaceous glands. Immunohistochemistry helps in differentiating the origin of the tumor, either eccrine or apocrine, positive immunoreactivity for proteins 15 and 24 and zinc-2 glycoprotein demonstrates evidence of apocrine differentiation, while positivity for CKs demonstrates eccrine differentiation [4]. Mari Kishibe and al [5] described a case of SCAP with a unique histopathology. The tumor was composed of basaloid cell proliferation interconnecting from the epidermis to the dermis. Ductal structures in the tumor were lined by club-shaped columnar cells with apical snouts. Numerous vacuolated cells with hyaline globule- like cytoplasmic inclusions were present among the columnar cells, the content of which was identified as sialomucin. Syringocystadenoma papilliferum is frequently seen in association with other benign adnexal lesions such as apocrine naevi, tubular apocrine adenomas, apocrine hidrocystomas, trichoadenomas, apocrine cystadenomas and clear cell syringomas [1]. Treatment of SCP is based on surgical excision, which is recommended for ulcerated forms or large size. When the location is unfavorable for surgical treatment, the CO2 laser therapy represents a good alternative [6]. Neurofibromatosis type 1 is one of the most frequent hereditary neurocutaneous disorders with a birth Syringocystadenoma papilliferum is presumably derived from pluripotent cells and differentiates toward eccrine and/or apocrine apparatuses [3]. There is also a possibility that SCAP can resemble lesions of the breasts deriving from the supernumerary mammary tissues from milk ridges. In some individuals those ridges may not regress completely during embryological development forming breast-like tissue [2]. The most frequent location is the head and neck. DISCUSSION Less frequently involved sites are face, chest, abdomen, arm, thighs, and perineum, location on vulva had only reported in few cases. The clinical presentation varies widely, making the clinical diagnosis of SCAP quite difficult. The tumor can present as a solitary nodule or greyish-brown papule with smooth and flat dome. Large nodules can ulcerate. Main dermoscopic features are exophytic papillary structures and polymorphous vessels1. Histopathologic appearance is uniform and characteristic the basis of diagnosis [3]. It typically shows varying degrees of papillomatosis along with cystic invaginations and malformed sebaceous glands. Immunohistochemistry helps in differentiating the origin of the tumor, either eccrine or apocrine, positive immunoreactivity for proteins 15 and 24 and zinc-2 glycoprotein demonstrates evidence of apocrine differentiation, while positivity for CKs demonstrates eccrine differentiation [4]. Mari Kishibe and al [5] described a case of SCAP with a unique histopathology. The tumor was composed of basaloid cell proliferation interconnecting from the epidermis to the dermis. Ductal structures in the tumor were lined by club-shaped columnar cells with apical snouts. Numerous vacuolated cells with hyaline globule- like cytoplasmic inclusions were present among the columnar cells, the content of which was identified as sialomucin. Syringocystadenoma papilliferum is frequently seen in association with other benign adnexal lesions such as apocrine naevi, tubular apocrine adenomas, apocrine hidrocystomas, trichoadenomas, apocrine cystadenomas and clear cell syringomas [1]. Treatment of SCP is based on surgical excision, which is recommended for ulcerated forms or large size. When the location is unfavorable for surgical treatment, the CO2 laser therapy represents a good alternative [6]. Neurofibromatosis type 1 is one of the most frequent h dit t di d ith bi th Figure 1: Café-au-lait macule with multiple freckling on the trunk. Figure 2: Pinkish to erythematous bordered elevated ulcerated nodule measuring 1 cm in diameter on the labia majora. Figure 2: Pinkish to erythematous bordered elevated ulcerated nodule measuring 1 cm in diameter on the labia majora. Figure 2: Pinkish to erythematous bordered elevated ulcerated nodule measuring 1 cm in diameter on the labia majora. Figure 3: Histological section (H and E × 20) showing papillary epithelial proliferation lined with a double layer of cuboidal and cylindrical cells. Figure 3: Histological section (H and E × 20) showing papillary epithelial proliferation lined with a double layer of cuboidal and cylindrical cells. cells confirming the diagnosis of SCAP (Fig. 3). CASE REPORT We report the case of a 36 years old patient, treated one year ago for a breast carcinoma in situ. With a family history of neurofibromatosis type 1 in the father, the dead brother and the sister. Physical examination showed many flat, uniformly 174 © Our Dermatol Online 2.2020 www.odermatol.com Figure 1: Café-au-lait macule with multiple freckling on the trunk. hamartoma, nuclear atypia or any signs of malignancy. No recurrence was noted with four months follow-up. CONCLUSION 4. Shah PA, Singh VS, Bhalekar S, Sudhamani S, Paramjit E. Syringocystadenoma papilliferum: A rare case report with review of literature. J Sci Soc. 2016;43:96-8. This case report contributes to extending NF1 associated tumors, and reconsider differential diagnoses of a chronic ulcerated lesion in the vulva. Our patient requires continuous and permanent monitoring to detect other neoplasms. 5. Mari K, Takeshi I, Ichiro T, Akemi IY. Intracytoplasmic eosinophilic inclusion bodies with embryonic folliculosebaceous-apocrine unit differentiation in syringocystadenoma papilliferum. J Cutan Pathol. 2018;45:923-6. 6. Bettioui A, Bouyahyaoui Y, Gallouj S. Syringocystadénome papillifère congénital. Ann Dermatol Vénéréol. 2012;139:647-51. 7. Rosenbaum T, Wimmer K. Neuroﬁ bromatosis type 1 (NF1) and Associated tumors. Klin Padiatr. 2014;226:309–15. REFERENCES 1. Mara L, Simonetta P, Caterina L, Borsari S, Persechino F, Argenziano G, et al. Dermoscopy of syringocystadenoma papilliferum. Austral J Dermatol. 2017;59:e59-e61. 2. Oleksandr S, Nirmala C, Frank L. Syringocysadenoma papilliferum of the vulva: a rarity in gynaecology. BMJ Case Rep. 2014;2014. pii: bcr2014203902. 3. Mohamed M, Belhadjali M, Hadhri R, Youssef M, Zili J. Clinical aspects of syringocystadenoma papilliferum. Our Dermatol Online. 2017;8:348. DISCUSSION There were no features of associated sebaceous 175 © Our Dermatol Online 2.2020 www.odermatol.com incidence of about 1/2500. It is caused by heterozygous germ-line mutations in the tumor suppressor gene NF1 which codes for neurofibromin, a negative regulator of the RAS proto-oncogene [7]. This makes NF1 patients at risk to develop various benign and malignant tumors. Authors [7] revealed that the range of NF1 associated tumors extends beyond nervous system and includes cancers of the lung, thyroid gland, skin, ovary and gastrointestinal tract. Our patient with the history of removed breast cancer, developed SCAP. To our knowledge, the association between NF1 and SCAP has not been described yet in the medical literature. Although this tumor had relatively benign course, rare cases of malignant transformation have been reported. patient(s) has/have given his/her/their consent for his/ her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed. © Our Dermatol Online 2.2020 Copyright by Iyda El Faqyr, 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. Source of Support: Nil, Conﬂ ict of Interest: None declared. Copyright by Iyda El Faqyr, 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. Source of Support: Nil Conﬂict of Interest: None declared Consent The examination of the patient was conducted according to the Declaration of Helsinki principles. The authors certify that they have obtained all appropriate patient consent forms. In the form the 176 © Our Dermatol Online 2.2020
https://openalex.org/W2809697215	https://europepmc.org/articles/pmc6336723?pdf=render	English	null	A Community-Wide Intervention Trial for Preventing and Reducing Frailty Among Older Adults Living in Metropolitan Areas: Design and Baseline Survey for a Study Integrating Participatory Action Research With a Cluster Trial	Journal of epidemiology	2,019	cc-by	8,207	Journal of Epidemiology Study Profile J Epidemiol 2019;29(2):73-81 A Community-Wide Intervention Trial for Preventing and Reducing Frailty Among Older Adults Living in Metropolitan Areas: Design and Baseline Survey for a Study Integrating Participatory Action Research With a Cluster Trial Satoshi Seino1, Akihiko Kitamura1, Yui Tomine1, Izumi Tanaka1, Mariko Nishi1, Kumiko Nonaka1, Yu Nofuji2, Miki Narita1, Yu Taniguchi1, Yuri Yokoyama1, Hidenori Amano1, Tomoko Ikeuchi1, Yoshinori Fujiwara1, and Shoji Shinkai3 1Research Team for Social Participation and Community Health, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan 2Japan Association for Development of Community Medicine, Tokyo, Japan 3Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Received May 22, 2017; accepted January 3, 2018; released online June 30, 2018 Journal of Epidemiology Study Profile J Epidemiol 2019;29(2):73-81 J Epidemiol 2019;29(2):73-81 A Community-Wide Intervention Trial for Preventing and Reducing Frailty Among Older Adults Living in Metropolitan Areas: Design and Baseline Survey for a Study Integrating Participatory Action Research With a Cluster Trial 1Research Team for Social Participation and Community Health, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan 2Japan Association for Development of Community Medicine, Tokyo, Japan 3Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan Received May 22, 2017; accepted January 3, 2018; released online June 30, 2018 Received May 22, 2017; accepted January 3, 2018; released online June 30, 2018 Address for correspondence. Satoshi Seino, PhD, Research Team for Social Participation and Community Health, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae, Itabashi, Tokyo 173-0015, Japan (e-mail: seino@tmig.or.jp). ABSTRACT This is an open access article distributed under the terms of Creative Commons Attribution Li permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. metropolitan areas is an important challenge. We have conducted community-based interventions for frailty prevention in hilly and mountainous and suburban areas in Japan.5–9 However, to the best of our knowledge, no previous study has reported eﬀective community-wide interventions or policies in Japanese metropolitan areas. DOI https://doi.org/10.2188/jea.JE20170109 HOMEPAGE http://jeaweb.jp/english/journal/index.html ABSTRACT Background: Preventing and reducing frailty is an important challenge for Japan in the next decade, especially in metropolitan areas. We launched a community-wide intervention trial (the Ota Genki Senior Project) in 2016 to develop eﬀective community- based strategies for frailty prevention in metropolitan areas. This report describes the study design and baseline survey. Background: Preventing and reducing frailty is an important challenge for Japan in the next decade, especially in metropolitan areas. We launched a community-wide intervention trial (the Ota Genki Senior Project) in 2016 to develop eﬀective community- based strategies for frailty prevention in metropolitan areas. This report describes the study design and baseline survey. Methods: This study is a community-wide intervention trial that integrates participatory action research into a cluster non- randomized controlled trial for adults aged 65 years or older living in Ota City, Tokyo. We allocated 3 of 18 districts to an intervention group and the other 15 to a control group. Using a mailed self-administered questionnaire, we conducted a baseline survey of 15,500 residents (8,000 and 7,500 in the intervention and control groups, respectively) from July through August 2016. In addition to socioeconomic status and lifestyle variables, we assessed frailty status (primary outcome) and physical, nutritional, and psychosocial variables (secondary outcomes). Based on the baseline ﬁndings, an intervention to improve outcomes will be implemented as participatory action research. Follow-up surveys will be conducted in the same manner as the baseline survey. baseline survey. Results: A total of 11,925 questionnaires were returned (76.9% response rate; 6,105 [76.3%] and 5,820 [77.6%] in the intervention and control groups, respectively), and 11,701 were included in the analysis (mean age, 74.3 [standard deviation, 5.5] years; 48.5% were men). y Results: A total of 11,925 questionnaires were returned (76.9% response rate; 6,105 [76.3%] and 5,820 [77.6%] in the intervention and control groups, respectively), and 11,701 were included in the analysis (mean age, 74.3 [standard deviation, 5.5] years; 48.5% were men). Conclusions: This study is expected to contribute to development of a prototype of a community-wide frailty prevention strategy, especially in metropolitan areas in Japan. Trial registration: UMIN Clinical Trials Registry (UMIN000026515). Conclusions: This study is expected to contribute to development of a prototype of a community-wide frailty prevention strategy, especially in metropolitan areas in Japan. Trial registration: UMIN Clinical Trials Registry (UMIN000026515). Key words: frailty; community-wide intervention; exercise; nutrition; social participation Copyright © 2018 Satoshi Seino et al. Journal of Epidemiology INTRODUCTION Frailty is a geriatric syndrome with multiple causes and contributors. It is characterized by diminished strength, endur- ance, and physiologic function, all of which increase the risks of increased dependence and death.1 Although frailty prevalence increases with age, especially in adults aged 75 or older,2 frailty can be prevented or reversed at an early stage. Therefore, preventing and reducing frailty is a major concern in geriatrics. Physical exercise, nutritional education, and active social participation should be the main targets of frailty prevention interventions.5 Recent randomized controlled trials (RCTs) showed that a multifactorial intervention comprising these factors eﬀectively reduced frailty and improved functional health.8,10–12 Physical exercise, nutritional education, and active social participation should be the main targets of frailty prevention interventions.5 Recent randomized controlled trials (RCTs) showed that a multifactorial intervention comprising these factors eﬀectively reduced frailty and improved functional health.8,10–12 The next step in such research is to determine how elements of a multifactorial program can be translated into practice in a community. To verify eﬀectiveness, it will be necessary to allocate communities rather than individuals. The estimated number of Japanese aged 75 years or older in 2025 (proportion of population, 18.1%) will be approximately 1.5 times that in 2010 (11.1%), because the postwar Baby- Boomer generation will reach age 75 by 2025.3,4 The phenomenon will be more remarkable in metropolitan areas.4 Thus, preventing and reducing frailty at the community level in The next step in such research is to determine how elements of a multifactorial program can be translated into practice in a community. To verify eﬀectiveness, it will be necessary to allocate communities rather than individuals. DOI https://doi.org/10.2188/jea.JE20170109 HOMEPAGE http://jeaweb.jp/english/journal/index.html DOI https://doi.org/10.2188/jea.JE20170109 HOMEPAGE http://jeaweb.jp/english/journal/index.html 73 Community-Wide Intervention Trial for Preventing=Reducing Frailty Community-Wide Intervention Trial for Preventing=Reducing Frailty Community-Wide Intervention Trial for Preventing=Reducing Frailty Community-Wide Intervention Trial for Preventing=Reducing Frailty that are generalizable but have enough ﬂexibility to be meaningful and are more likely to be successful locally.15 The trial is registered in the UMIN Clinical Trials Registry (UMIN000026515). On the basis of past studies, we launched the community-wide intervention trial for preventing and reducing frailty in Ota City, Tokyo (the Ota Genki Senior Project), in 2016. The aims of this study are to develop and examine the eﬀectiveness of a social mechanism (ie, intervention content) that postpones frailty and to determine the requirements for transferring the intervention to other communities (transferability). INTRODUCTION Using this process, we aim to develop a prototype of a frailty prevention strategy for metropolitan areas in Japan. This report describes the study design and baseline proﬁle of the participants. Study design The Ota Genki Senior Project is a community-wide intervention study integrating participatory action research (PAR)13,14 into cluster non-RCTs for individuals aged 65 years or older living in Ota City, Tokyo, Japan. Although a multi-pronged approach is eﬀective in postponing frailty,8,10–12 standardized interventional approaches are less successful at the community level because of inherent diﬀerences between communities, such as human and regional resources and organizational structure. Moreover, translating a “one-size-ﬁts-all” intervention from one community to another may be diﬃcult for the same reasons. Ota City is the southernmost of the 23 special wards of Tokyo. On August 1, 2016 it had a population of 716,645 (357,748 males and 358,897 females), 162,443 (71,481 men and 90,962 women) of whom were aged 65 years or older. The proportion of elders to the total population was 22.7%, which is slightly higher than the mean for the other special wards (21.1% as of January 2016).17 The area of Ota City (60.66 km2) is the largest of the special wards, although Tokyo International Airport accounts for approximately a quarter. The population density was 11,814 persons=km2 (habitable area: approximately 15,750 persons=km2). To overcome this problem, an implementation research design integrating PAR into RCTs was proposed as a new approach for studying interventions in healthcare settings.15 PAR is a design that brings together researchers and stakeholders (eg, residents, civil groups, professionals, company, and government) in a collaborative eﬀort to address issues in speciﬁc systems.13–16 It is a collaborative, cyclical (analogous to “plan–do–check–act”), and reﬂective inquiry design that focuses on problem solving and understanding the eﬀect of an intervention as part of the research process.15 Study setting In response to a request from Ota City, we proposed and began a 3-year collaborative research project. In October 2011, we started work for 3 years on “Development of a Community-based Comprehensive System for Prevention of Frailty in Late Life.”9 The intent was to use high-quality evidence to create a frailty prevention theory and to use this theory for community planning aimed at postponing frailty through cooperation with community residents and government oﬃcials in Yabu City, Hyogo Prefecture (a hilly and mountainous area) and Hatoyama Town, Saitama Prefecture (a suburban area). On the basis of these premises, we hope to construct a model for frailty prevention in greater Tokyo. Allocation As shown in Figure 1, there are 18 administrative districts within Ota City, and the eligibility criterion for clusters applies to all districts. Normally, the intervention district should be selected randomly. However, the results of initial meetings with city employees and community diagnoses based on previous results of Ota City surveys of actual conditions indicated that east and west We attempt to expand this design by integrating PAR into cluster non-RCTs. Hence, we can design interventional trials Tokyo International Airport Ota City District A District B District C Figure 1. Geographical location of study areas (Ota City, Tokyo, Japan) District C District B District A Ota City Figure 1. Geographical location of study areas (Ota City, Tokyo, Japan) 74 j J Epidemiol 2019;29(2):73-81 Seino S, et al. Seino S, et al. Ota City diﬀered greatly in characteristics such as regional sources, participation rates for resident associations=neighbor- hood associations, and characteristics such as main cause of death. Therefore, we chose a non-randomized design and selected intervention districts from both east and west Ota City. Second, to discuss and develop intervention content based on the baseline ﬁndings, a monthly “Community Consultation” is held in each intervention district. Members of the Community Consultation include stakeholders, such as residents, pro- fessionals (caseworkers, care managers, mental health welfare professionals, managerial dieticians, exercise instructors, and others), and members of Community Support Service Centers, social welfare corporations, employment service centers for older people, companies, research institutions, and government (city employees) (“Plan 2”). Next, arrangements were made at the Ward Oﬃce to select the intervention districts. District A (population, 38,301; elderly population, 8,797 [23.0%]), which is located in the east, and districts B and C (considered as one cluster; population, 47,183; elderly population, 10,914 [23.1%]), which are located in the west, were allocated to the intervention group; the other 15 districts were allocated to the control group. Statistical data on rates of long-term care insurance certiﬁcation for Ota City, as of 2016, showed that the rates in districts A, B, and C were the second, ﬁfth, and sixth highest, respectively, of the 18 districts, and we thus considered that these districts are suitable as intervention districts. Third, on the basis of the plan developed in the Community Consultation, interventions that focus on improving dietary variety and physical and social activities are implemented as PAR for each intervention district (“Do”). Allocation The intervention contents are 1) to establish resident-oriented activities related to frailty prevention (ie, physical exercise, nutrition, and=or social participation), and 2) to improve the community environment (ie, communicating the importance of exercise activities, dietary variety, and active social participation by cooperating organ- izations and implementing these as part of existing eﬀorts). Districts B and C neighbor each other, and their elderly populations (district B, 5,514; district C, 5,400) are smaller than that of district A (elderly population, 8,797). In addition, discussions with the directors of the Community Support Service Centers revealed that there were few community resources, such as shopping centers, in district C, and that residents of district C also use shopping centers and sports clubs located in district B. Furthermore, the directors of Community Support Service Centers in both districts wanted to carry out joint regional activities between districts B and C in the future. Therefore, we decided to start the project by placing districts B and C in the same cluster. p g p g ) Currently, in district A, regular group meals and walking groups are planned. A lecture series (lecture + exercise practice) is oﬀered in all 10 neighborhood associations. Additionally, the “district A squat challenge” is being held using information communication equipment to disseminate the squatting exercise. In district B, regular walking-pole groups are planned in all ﬁve neighborhood associations. Additionally, nutritional lectures and group meals are planned in various locations in the district, such as shopping malls. We are also developing software applications to visualize walking practices and daily food intakes. In district C, in accordance with the concept of “community building that makes you want to go out,” the members are studying a plan to spread awareness of frailty prevention for participants, by holding regular events that include a walk around the district followed by a group meal. Currently, in district A, regular group meals and walking groups are planned. A lecture series (lecture + exercise practice) is oﬀered in all 10 neighborhood associations. Additionally, the “district A squat challenge” is being held using information communication equipment to disseminate the squatting exercise. In district B, regular walking-pole groups are planned in all ﬁve neighborhood associations. Additionally, nutritional lectures and group meals are planned in various locations in the district, such as shopping malls. We are also developing software applications to visualize walking practices and daily food intakes. Outcomes As shown in Table 1, outcome measures include frailty status (primary outcome) and physical and psychosocial variables and dietary variety (secondary outcomes), in addition to socio- economic status and lifestyle variables. Procedures for assessing the primary and secondary outcome measures and additional measures are detailed in eMaterial 1. We used both stratiﬁed sampling of four groups classiﬁed by age group (65–74 and 75–84 years) and sex (men and women) and random sampling strategies for recruitment of study participants from the 18 districts. Individuals with long-term care insurance certiﬁcation and those admitted to hospitals or residing in nursing homes were excluded. The target population for sampling was 15,500 residents (8,000 and 7,500 people in the intervention and control groups, respectively), which is equivalent to approximately one-tenth of the elderly population of Ota City. In the intervention group, 4,000 individuals (1,000 from each of the four stratiﬁed groups) from district A and 2,000 individuals (500 from each of the four stratiﬁed groups) each from districts B and C were recruited; thus, the questionnaire was distributed to approximately one out of every two residents. In the control group, 500 individuals (125 from each of the four stratiﬁed groups) from each of the other 15 districts were recruited. Community-Wide Intervention Trial for Preventing=Reducing Frailty Posters and recipes are created to raise awareness of the importance of dietary variety and are posted in shopping locations and public facilities in intervention districts. In the control group, usual health practices are continued. will be repeated every 3 years. Follow-up questionnaires will be mailed to respondents to the baseline survey. To improve assessment of individual-level and community-level eﬀects, we will add an additional sample of randomly extracted adults of the same sex and age group as those who dropped out at each follow-up survey. Fourth, the intervention’s eﬀects will be assessed by means of a 2-year follow-up survey, and the intervention content will be revised as needed (“Check”). Moreover, the requirements necessary for transfer of the intervention to other communities will be identiﬁed (“Consideration of Transferability”). In concrete terms, we will create a list of all intervention content implemented in the three intervention districts after the end of the 2-year follow-up survey. Using this list, we plan to host a core committee comprising ourselves, city employees, and directors of the Community Support Service Centers of each district to discuss implementation of the interventions in other districts. As in many previous studies,20–22 it may be diﬃcult to produce outcome changes in only 2 years, and long-term follow-up may be required. We plan to repeat follow-up surveys every 3 years after the 2-year follow-up. Moreover, we plan to use a stepped- wedge design23 to expand the intervention areas within the 18 districts, apart from districts A, B, and C. Diﬀerences between the ﬁrst intervention group (districts A, B, and C), districts included in the following intervention areas, and control districts will be examined by every follow-up survey. Furthermore, the previous logic model suggests that change would be induced by the intervention in the following order: awareness, knowledge, belief, intention, and ﬁnally action,20 and we will identify the stage we have reached at each follow-up survey. Finally, we will examine whether the created social mechanism decreased the long-term care insurance certiﬁcation rate and=or extended healthy life expectancy. These steps are based on the PAR framework.18 Finally, mortality and long-term care insurance certiﬁcation after the baseline survey are expected to be investigated in future exploratory studies. Secondary outcome measures Physical activity and physical function Physical activity and physical function Engaging in any exercise and muscle-strengthening activities 1 or more days=week and engaging in 150 minutes=week or more of walking were evaluated.20,26–28 Physical function was assessed using the Motor Fitness Scale.29,30 Mobility limitations were identiﬁed through self-reported diﬃculty in walking one-quarter of a mile (0.4 km) or climbing 10 steps without resting.31,32 Dietary variety On the basis of available data from the Yabu Cohort Study, the estimated prevalence of frailty at baseline was 27.5%,7 and the estimated intra-cluster correlation coeﬃcient (ICC) was 0.0112 (ICC was unpublished). From these data, we assumed the prevalence of frailty in Ota City as 25%, with an ICC of 0.01. Sample size was calculated using the chi-square test at the individual level, a two-sided 5% signiﬁcance level, and a power of 80% to detect a 6% diﬀerence in change in the prevalence of frailty between the intervention and control groups, without considering the design eﬀect (DE) by cluster trial.19 Thus, samples of 748 persons were obtained for the intervention and control group (total: 1,496). When we assume a ﬁnal sample with a 50% response rate at baseline, the estimated DE is 5.29. The product of the obtained sample size of 748 in the intervention and control groups and estimated DE of 5.29 is 3,957. Therefore, we considered that recruiting 8,000 participants in the intervention group and 7,500 participants in the control group would retain the expected statistical power throughout follow-up. Dietary Variety Score (DVS, range 0–10)33,34 and Food Frequency Score (FFS, range 0–30)34 were assessed with a self-administered questionnaire. DVS was treated as both a continuous and dichotomous (score of ≥4) variable. Psychosocial function Self-rated health, depressive mood, well-being, frequency of outing, social isolation, and cognitive and structural social capital were determined by analyzing responses to a commonly used self-administered questionnaire.35–39 Baseline and follow-up surveys This baseline survey was conducted to collect baseline data for the intervention study, to clarify the current living conditions of older adults living in Ota City, and to serve as material for intervention content. Primary outcome measures The primary outcomes are Check-List 15 (CL15) score and frailty status (frailty deﬁned as a CL15 score ≥4).5,24,25 CL15 score was treated as both a continuous and dichotomous (prevalence of frailty) variable in the analysis. 76 j J Epidemiol 2019;29(2):73-81 Overview of the intervention Figure 2 shows the research roadmap of the Ota Genki Senior Project. The ﬁrst step, conducting the baseline survey, was performed in July and August 2016. The data were used to analyze health challenges regarding exercise, nutrition, and=or social activity that should be addressed in each intervention district (“Plan 1”). In district C, in accordance with the concept of “community building that makes you want to go out,” the members are studying a plan to spread awareness of frailty prevention for participants, by holding regular events that include a walk around the district followed by a group meal. Preventing or reducing frailty Improving physical activity, dietary variety, psychosocial variables Follow-up surveys will be conducted. 2016.7-8 From 2016.12 Social activity Dietary variety Physical activity Middle-term aims Long-term aims Actual condition survey and analysis of health challenges Baseline survey was conducted for a sample of 15500 participants, and health challenges for each intervention cluster were analyzed. Plan 1 Identification and analysis of problems requiring solutions in the community Plan 2 Making action plan and team building for solutions Community Consultation On the basis of the survey findings, intervention content will be discussed and implemented by stakeholders. Do Implementation of actions based on the plan Check Process and outcome evaluation and revision of intervention content Consideration of transferability Phase 1 (3 years) Phase 2 Process as the PAR18 Multipronged intervention at the community level Approaches to population and community environment Extending healthy life expectancy Reducing long-term care insurance certification rate 1) Establishing resident-oriented activities related to frailty prevention (i.e., physical exercise, nutrition, and/or social participation) 2) Improving the community environment (i.e., communicating the importance of exercise activities, dietary variety, and active social participation by cooperating organizations and implementing these as part of existing efforts). Figure 2. Research roadmap of the Ota Genki Senior Project. PAR, participatory action research. Extending healthy life expectancy aims Extending healthy life expectancy Reducing long-term care insurance certification rate J Epidemiol 2019;29(2):73-81 j Community-Wide Intervention Trial for Preventing=Reducing Frailty RESULTS Figure 3 shows a ﬂow diagram of the study’s progress. Of the 15,500 questionnaires distributed, 11,925 were returned (76.9% response rate; 6,105 [76.3%] and 5,820 [77.6%] of the intervention and control groups, respectively). After excluding 79 questionnaires from respondents who did not actually live in the city, 38 questionnaires that were almost or completely blank, 19 questionnaires with missing identiﬁcation labels, 22 questionnaires that were completed by someone other than the participant, and 66 questionnaires from hospital inpatients and nursing home residents (total 224), 11,701 were ultimately identiﬁed as the study population and included in the analysis (75.5% valid response rate; 6,009 [75.1%] and 5,692 [75.9%] of the intervention and control groups, respectively). Table 2 shows the baseline characteristics of the study population. Compared with participants in the control group, those in the intervention group were less likely to be living alone, had less current smoking, knee pain, depressive mood, and social isolation, were more likely to be married and have social participation more than once a week, and had higher educational level, equivalent income, and DVS and FFS scores. The multiple- comparison test showed that frailty status and almost all secondary outcome measures were signiﬁcantly worse in district A and better in districts B and C than in the control group. No signiﬁcant diﬀerence was observed in any other measure. gave informed consent. A statement attached to the questionnaire explained the purpose of the study, the voluntary nature of participation, and a promise of anonymity in the analysis. Returning the questionnaire was viewed as consent to participate in the study. . Summary of items surveyed in the Ota Genki Senior Project at baseline, 2016 Summary of items surveyed in the Ota Genki Senior Project at baseline, 2016 As primary analyses, we will use generalized linear mixed- eﬀects models (GLMM) to compare changes in all outcomes between the intervention group (3 districts) and control group (15 districts). Each outcome will be deﬁned as a dependent variable. Group, time (baseline and follow-up), and their interaction will be deﬁned as ﬁxed factors, and the districts where respondents live will be deﬁned as a random factor. Sex, age, body mass index, chronic disease, socioeconomic status, and lifestyle at baseline, and the baseline value of each outcome will be deﬁned as covariates. The intervention eﬀect and its 95% conﬁdence interval will be calculated as an estimate of the mean diﬀerences in changes between groups, after controlling for covariates. Project at baseline, 2016 Primary outcome measures Check-List 15 and frailty status Secondary outcome measures Physical activity and physical function Exercise habits International Physical Activity Questionnaire Short-Form Motor Fitness Scale Mobility limitations Dietary variety Dietary Variety Score Food Frequency Score Psychosocial function Self-rated health Five-item version of the Geriatric Depression Scale WHO-5 Well-Being Index Frequency of outing Social isolation Cognitive social capital (trust in neighbors, etc) Structural social capital (social participation, etc) Additional measures Age Sex Living arrangement Marital status Years of residence in neighborhood area Socioeconomic status (education, household income, etc) Employment status History of physician-diagnosed diseases and chronic musculoskeletal pain Body mass index (self-rated height and weight) Drinking and smoking Sleep duration, diﬃculty falling asleep, and quality of sleep Pet breeding situation Information and communication technology use Tokyo Metropolitan Institute of Gerontology Index of Competence Number of meals Eating alone Level of happiness Social network, social support, and neighbor relationships WHO, World Health Organization. Ethical considerations The study protocol was developed in accordance with the guidelines proposed in the Declaration of Helsinki and was approved by the Ethics Committee of the Tokyo Metropolitan Institute of Gerontology (approved June 1, 2016). All participants After conducting the baseline survey, a 2-year follow-up questionnaire survey is planned. After that, follow-up surveys 76 j J Epidemiol 2019;29(2):73-81 Seino S, et al. Seino S, et al. Seino S, et al. Statistical analyses All data in the text and tables are presented as means (standard deviations) or proportions. We used the unpaired t test, Mann- Whitney U test, or chi-square test to compare baseline characteristics between groups. Moreover, to compare district A, districts B and C with the control, we used analysis of variance, the Kruskal-Wallis test, or chi-square test. Post-hoc multiple-comparison testing with Bonferroni adjustment was performed for primary and secondary outcome measures. We calculated the ICCs for primary and secondary outcome measures as follows: The results of analysis of the additional measures are shown in eTable 1. As compared with participants in the control group, those in the intervention group were more likely to eat with others and to be working and had more interactions with neighbors. No signiﬁcant diﬀerence was observed in any other measure. Primary outcome measures As secondary analyses, we will use a GLMM to compare each intervention district to the control group for primary and secondary outcomes, in the same manner. Furthermore, subgroup analyses will be used to detect signiﬁcant diﬀerences in the intervention eﬀect, after stratiﬁcation by sex and=or age group (65–74 and 75–84 years). DISCUSSION We described the study design and baseline characteristics of participants in the present study, which was launched in 2016. To the best of our knowledge, this is the ﬁrst intervention study for community-wide frailty prevention in a metropolitan area. Dietary variety and some variables related to psychosocial and socioeconomic status were better for participants in the intervention group than for those in the control group. Further multiple-comparison testing showed that socioeconomic status, We described the study design and baseline characteristics of participants in the present study, which was launched in 2016. To the best of our knowledge, this is the ﬁrst intervention study for community-wide frailty prevention in a metropolitan area. ICC ¼ ðBMS WMSÞ=ðBMS þ ½K 1WMSÞ; . Summary of items surveyed in the Ota Genki Senior Project at baseline, 2016 Project at baseline, 2016 Primary outcome measures Check-List 15 and frailty status Secondary outcome measures Physical activity and physical function Exercise habits International Physical Activity Questionnaire Short-Form Motor Fitness Scale Mobility limitations Dietary variety Dietary Variety Score Food Frequency Score Psychosocial function Self-rated health Five-item version of the Geriatric Depression Scale WHO-5 Well-Being Index Frequency of outing Social isolation Cognitive social capital (trust in neighbors, etc) Structural social capital (social participation, etc) Additional measures Age Sex Living arrangement Marital status Years of residence in neighborhood area Socioeconomic status (education, household income, etc) Employment status History of physician-diagnosed diseases and chronic musculoskeletal pain Body mass index (self-rated height and weight) Drinking and smoking Sleep duration, diﬃculty falling asleep, and quality of sleep Pet breeding situation Information and communication technology use Tokyo Metropolitan Institute of Gerontology Index of Competence Number of meals Eating alone Level of happiness Social network, social support, and neighbor relationships WHO, World Health Organization. WHO, World Health Organization. ICC ¼ ðBMS WMSÞ=ðBMS þ ½K 1WMSÞ; where BMS is the between-cluster mean square, WMS is the within-cluster mean square, and K is the average number of respondents per cluster.19,20 An α of 0.05 was considered to indicate statistical signiﬁcance, and all statistical analyses were performed using Stata 14.0 (Stata Corp, College Station, TX, USA). Dietary variety and some variables related to psychosocial and socioeconomic status were better for participants in the intervention group than for those in the control group. Further multiple-comparison testing showed that socioeconomic status, J Epidemiol 2019;29(2):73-81 77 Community-Wide Intervention Trial for Preventing=Reducing Frailty Assessed for eligibility (all 18 administrative districts in Ota City) Allocated to control group (15 districts) Allocated to intervention group (3 districts) District A Districts B and C Baseline survey: questionnaires mailed to 15,500 (8,000 in the intervention group and 7,500 in the control group) participants 500 in each district selected using stratified and random sampling (n=7,500) 2-year follow-up survey: Follow-up questionnaires will be mailed to respondents to baseline survey Analyses with intention-to-treat and per-protocol bases Intervention by participatory action research at the community level 4,000 selected using stratified and random sampling (n=4,000) 2,000 in each district selected using stratified and random sampling (n=4,000) Survey responses: 3,098 (77.5%) Excluded: 48 Included in analysis: 3,050 (76.3%) Analysis Follow-up Allocation Enrolment Survey responses: 3,007 (75.2%) Excluded: 48 Included in analysis: 2,959 (74.0%) Survey responses: 5,820 (77.6%) Excluded: 128 Included in analysis: 5,692 (75.9%) Usual health practices Figure 3. Flow diagram of progress in the Ota Genki Senior Project Allocation Enrolment Assessed for eligibility (all 18 administrative districts in Ota City) Allocated to control group (15 districts) 500 in each district selected using stratified and random sampling (n=7,500) 2,000 in each district selected using stratified and random sampling (n=4,000) aseline survey: questionnaires mailed to 15,500 (8,000 in the intervention group and 7,500 in the control group) participants Survey responses: 5,820 (77.6%) Excluded: 128 Included in analysis: 5,692 (75.9%) Figure 3. Flow diagram of progress in the Ota Genki Senior Project frailty status, and physical, nutritional, and psychosocial variables were notably worse in district A than in districts B and C and the control group, regardless of age. For this reason, district A is positioned as a “typical model area” for solving health challenges. Because interaction with neighbors is active in district A (eTable 1), the word-of-mouth information dissemination strategy using this feature was considered eﬀective. ICC ¼ ðBMS WMSÞ=ðBMS þ ½K 1WMSÞ; Based on proportionate universalism (that is, applying a universal intervention with increased intensity in groups with greater need),40,41 intensive intervention is needed for district A in particular within the intervention group. communities is labor- and cost-intensive and may not be realistic. Therefore, we will create a project content list (eg, regular walking group, group meals, and=or printing for the creation and distribution of pamphlets) that will explicitly indicate the processes implemented in them and how much funding was needed for each component. Using this list, we believe it will be easier for other communities to select and implement the components of this study. This is an eﬀectiveness study that attempts to disseminate and implement its ﬁndings,8 the eﬀects of which were examined in a RCT (eﬃcacy study). Combining PAR and cluster non-RCTs, as in the present study, increases the ability to translate research ﬁndings into general practice in other communities. The process and results of this study can contribute to future dissemination and implementation research and to a Community-based Integrated Care System,42 which is promoted in Japan. Socioeconomic status, frailty status, and secondary outcome measures were better in districts B and C than in the control group, regardless of age. In addition, the utilization rate of information and communication technology (ICT), such as smartphones, computers, and internet was prohibitively high (eTable 1). In the next decade, the aﬃnity of the elderly for ICT is expected to progressively increase. Therefore, we positioned districts B and C as a model area for development of ICT equipment and applications that can be used for frailty prevention (promoting physical exercise, diverse food intake, and active social participation). Our study has some limitations and concerns. First, the self- administered questionnaire used for outcome measurement may be participant to recall bias. Second, complete prevention of potential contamination is diﬃcult because this study is performed as part of an administrative policy. Similarly, public relations at public facilities or shopping malls in intervention districts will be eﬀective, but it is diﬃcult to prevent contamination as new people enter the intervention district. Third, substantial attrition during the follow-up surveys and a large diﬀerence between the intervention and control groups will lead to attrition bias and loss of statistical power. 78 j J Epidemiol 2019;29(2):73-81 ICC ¼ ðBMS WMSÞ=ðBMS þ ½K 1WMSÞ; Because the Continuation of community-wide intervention studies and resident-oriented activities will require an annual budget of more than 10 million yen, especially when both the intervention and follow-up surveys are conducted on this scale. However, implementation of all the content of this research in other 78 j J Epidemiol 2019;29(2):73-81 Seino S, et al. able 2. Baseline characteristics of participants selected with stratiﬁed and random sampling, by age and sex (Ota Genki Senior Project in 2016) I i P l acteristics of participants selected with stratiﬁed and random sampling, by age and sex (Ota Genki Senior Project acteristics of participants selected with stratiﬁed and random sampling, by age and sex (Ota Genki Senior Project Table 2. ICC ¼ ðBMS WMSÞ=ðBMS þ ½K 1WMSÞ; Baseline characteristics of participants selected with stratiﬁed and random sampling, by age and sex (Ota Genki Senior Project in 2016) Variables Intervention group Control group P-value ICCd All District A Districts B and C Intervention vs controlb A vs B & C vs controlc Number of clusters 3 1 2 15 Number of residents 85,484 38,301 47,183 631,161 Number of residents aged 65 or older 19,711 8,797 10,914 142,732 Number of analyzed participants (eligible response rate) 6,009 (75.1) 3,050 (76.3) 2,959 (74.0) 5,692 (75.9) Age, years, mean (SD) 74.3 (5.4) 74.1 (5.3) 74.4 (5.5) 74.3 (5.5) 0.93 0.08 65–74, n (%) 2,848 (47.4) 1,468 (48.1) 1,380 (46.6) 2,669 (46.9) 0.58 0.44 75–84, n (%) 3,161 (52.6) 1,582 (51.9) 1,579 (53.4) 3,023 (53.1) Male, n (%) 2,902 (48.3) 1,465 (48.0) 1,437 (48.6) 2,768 (48.6) 0.72 0.86 Living alone, n (%) 1,136 (19.3) 628 (21.1) 508 (17.5) 1,242 (22.4) <0.001 <0.001 Marital status, n (%) <0.001 <0.001 Married 4,032 (68.8) 1,979 (66.9) 2,053 (70.7) 3,598 (64.8) Widowed or Divorced 1,460 (24.9) 787 (26.6) 673 (23.2) 1,494 (26.9) Never married 367 (6.3) 190 (6.4) 177 (6.1) 460 (8.3) Education, n (%) <0.001 <0.001 Junior high school graduate 1,496 (24.9) 1,207 (39.6) 289 (9.8) 1,414 (24.8) High school graduate 2,122 (35.3) 1,207 (39.6) 915 (30.9) 2,287 (40.2) Junior college=vocational college graduate 691 (11.5) 231 (7.6) 460 (15.5) 637 (11.2) College=graduate school graduate 1,457 (24.2) 238 (7.8) 1,219 (41.2) 1,158 (20.3) Other=unknown 243 (4.0) 167 (5.5) 76 (2.6) 196 (3.4) Equivalent income, n (%) <0.001 <0.001 ≥4.0 million yen 1,191 (19.8) 344 (11.3) 847 (28.6) 914 (16.1) 2.5–3.99 million yen 1,385 (23.0) 613 (20.1) 772 (26.1) 1,401 (24.6) 1.0–2.49 million yen 1,811 (30.1) 1,171 (38.4) 640 (21.6) 1,819 (32.0) <1.0 million yen 419 (7.0) 290 (9.5) 129 (4.4) 390 (6.9) Unknown 1,203 (20.0) 632 (20.7) 571 (19.3) 1,168 (20.5) Alcohol drinking status, n (%) 0.24 <0.001 Current 3,288 (55.4) 1,599 (53.2) 1,689 (57.7) 3,037 (54.0) Never or former 2,645 (44.5) 1,408 (46.8) 1,237 (42.3) 2,584 (46.0) Smoking status, n (%) 0.036 <0.001 Current 702 (11.9) 412 (13.9) 290 (9.9) 748 (13.4) Never or former 5,192 (88.0) 2,562 (86.2) 2,630 (90.0) 4,823 (86.6) Body mass index (kg=m2), mean (SD) 22.7 (3.2) 23.1 (3.2) 22.4 (3.1) 22.7 (3.2) 0.35 <0.001 Number of chronic diseases, n (%)a 0.78 0.59 0 1,391 (25.7) 675 (24.8) 716 (26.6) 1,294 (25.5) 1 1,767 (32.6) 898 (33.0) 869 (32.3) 1,691 (33.3) 2+ 2,257 (41.7) 1,150 (42.2) 1,107 (41.1) 2,097 (41.3) Musculoskeletal pain, n (%) Shoulder 1,213 (22.1) 654 (23.4) 559 (20.7) 1,198 (23.1) 0.22 0.025 Low back 2,228 (39.9) 1,186 (42.0) 1,042 (37.8) 2,095 (39.8) 0.90 0.007 Knee 1,762 (31.8) 998 (35.4) 764 (28.0) 1,775 (34.0) 0.016 <0.001 Primary outcome measures Frailty (CL15 score ≥4), n (%) 1,248 (23.0) 726 (27.0) 522 (19.1)+ 1,243 (24.1)+,++ 0.18 <0.001 0.0072 CL15 score (0–15), mean (SD) 2.3 (2.1) 2.5 (2.1) 2.1 (2.0)+ 2.4 (2.1)+,++ 0.32 <0.001 0.0085 Secondary outcome measures Physical activity and physical function Engaging in any exercise more than once a week, n (%) 4,341 (73.9) 2,061 (69.5) 2,280 (78.3)+ 4,095 (73.9)+,++ 0.98 <0.001 0.0066 Engaging in ≥150 minutes=week of walking, n (%) 3,045 (70.2) 1,389 (66.6) 1,656 (73.4)+ 2,958 (71.9)+ 0.08 <0.001 0.0051 Engaging in muscle-strengthening activities more than once a week, n (%) 533 (9.1) 200 (6.7) 333 (11.4)+ 511 (9.2)+,++ 0.78 <0.001 0.0036 Motor Fitness Scale (0–14), mean (SD) 10.6 (3.3) 10.3 (3.3) 10.9 (3.2)+ 10.5 (3.3)+,++ 0.32 <0.001 0.0081 Low physical function (score ≤11 for men, ≤9 for women), n (%) 2,149 (39.5) 1,184 (43.5) 965 (35.5)+ 2,086 (40.6)+,++ 0.25 <0.001 0.0061 Mobility limitation, n (%) 1,742 (29.4) 1,051 (35.1) 691 (23.6)+ 1,697 (30.3)+,++ 0.29 <0.001 0.0116 Dietary intake Dietary Variety Score (0–10), mean (SD) 3.2 (2.2) 2.9 (2.2) 3.5 (2.2)+ 3.1 (2.2)+,++ 0.040 <0.001 0.0175 Score ≥4, n (%) 2,237 (41.5) 937 (35.3) 1,300 (47.5)+ 2,009 (39.3)+,++ 0.021 <0.001 0.0123 Food Frequency Score (0–30), mean (SD) 18.0 (5.2) 17.2 (5.3) 18.8 (5.0)+ 17.7 (5.2)+,++ 0.002 <0.001 0.0185 Psychosocial function Self-rated health (excellent to good), n (%) 4,514 (80.6) 2,160 (76.7) 2,354 (84.6)+ 4,298 (80.9)+,++ 0.69 <0.001 0.0083 Depressive mood (GDS-5 score ≥2), n (%) 1,877 (33.7) 1,070 (38.5) 807 (29.0)+ 1,889 (35.8)++ 0.021 <0.001 0.0115 Well-being (WHO-5 Well-Being Index: 0–25), mean (SD) 15.3 (6.0) 14.5 (6.3) 16.1 (5.6)+ 15.2 (6.0)+,++ 0.37 <0.001 0.0134 Outing more than once a day, n (%) 4,366 (73.8) 2,220 (74.2) 2,146 (73.3) 4,202 (75.0) 0.14 0.25 0.0009 Social isolation, n (%) 1,632 (27.8) 951 (32.1) 681 (23.4)+ 1,664 (29.9)++ 0.013 <0.001 0.0060 Trust in neighbors, (agree or tend to agree), n (%) 4,414 (77.5) 2,113 (74.0) 2,301 (81.0)+ 4,129 (76.3)+,++ 0.16 <0.001 0.0055 Social participation more than once a month, n (%) 2,387 (45.5) 996 (38.1) 1,391 (52.8)+ 2,141 (43.3)+,++ 0.027 <0.001 0.0163 CL15, Check-List 15; GDS-5, 5-item version of the Geriatric Depression Scale; ICC, intracluster correlation coeﬃcient; SD, standard deviation; WHO, World Post-hoc multiple-comparison testing with Bonferroni adjustment was performed for primary and secondary outcome measures: +P < 0.017 versus district A; ++P < 0.017 versus districts B and C. CL15, Check-List 15; GDS-5, 5-item version of the Geriatric Depression Scale; ICC, intracluster correlation coeﬃcient; SD, stan Health Organization. g um of the presence of hypertension, hyperlipidemia, cardiovascular disease, cerebrovascular disease, and diabetes mellitus. Check-List 15; GDS-5, 5-item version of the Geriatric Depression Scale; ICC, intracluster correlation coeﬃcient; SD, standard devi O i ti REFERENCES 23. Mdege ND, Man MS, Taylor Nee Brown CA, et al. Systematic review of stepped wedge cluster randomized trials shows that design is particularly used to evaluate interventions during routine implementation. J Clin Epidemiol. 2011;64:936–948. 1. Morley JE, Vellas B, van Kan GA, et al. Frailty consensus: a call to action. J Am Med Dir Assoc. 2013;14:392–397. 2. Shimada H, Makizako H, Doi T, et al. Combined prevalence of frailty and mild cognitive impairment in a population of elderly Japanese people. J Am Med Dir Assoc. 2013;14:518–524. 24. 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https://openalex.org/W2132820424	https://link.springer.com/content/pdf/10.1007/s11671-010-9616-z.pdf	English	null	Fabrication of vertically aligned Pd nanowire array in AAO template by electrodeposition using neutral electrolyte	Nanoscale research letters	2,010	cc-by	4,292	Nanoscale Res Lett (2010) 5:1137–1143 DOI 10.1007/s11671-010-9616-z Nanoscale Res Lett (2010) 5:1137–1143 DOI 10.1007/s11671-010-9616-z NANO EXPRESS Fabrication of vertically aligned Pd nanowire array in AAO template by electrodeposition using neutral electrolyte Nevin Tas¸altın • Sadullah O¨ ztu¨rk • Necmettin Kılınc¸ • Hayrettin Yu¨zer • Zafer Ziya O¨ ztu¨rk Received: 17 December 2009 / Accepted: 16 April 2010 / Published online: 1 May 2010 The Author(s) 2010. This article is published with open access at Springerlink.com Abstract A vertically aligned Pd nanowire array was successfully fabricated on an Au/Ti substrate using an anodic aluminum oxide (AAO) template by a direct voltage electrodeposition method at room temperature using diluted neutral electrolyte. The fabrication of Pd nanowires was controlled by analyzing the current–time transient during electrodeposition using potentiostat. The AAO template and the Pd nanowires were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) meth- ods and X-Ray diffraction (XRD). It was observed that the Pd nanowire array was standing freely on an Au-coated Ti substrate after removing the AAO template in a relatively large area of about 5 cm2, approximately 50 nm in diameter and 2.5 lm in length with a high aspect ratio. The nucle- ation rate and the number of atoms in the critical nucleus were determined from the analysis of current transients. Pd nuclei density was calculated as 3.55 9 108 cm-2. Usage of diluted neutral electrolyte enables slower growing of Pd nanowires owing to increase in the electrodeposition potential and thus obtained Pd nanowires have higher crystallinity with lower dislocations. In fact, this high crystallinity of Pd nanowires provides them positive effect for sensor performances especially. N. Tas¸altın S. O¨ ztu¨rk N. Kılınc¸ Z. Z. O¨ ztu¨rk (&) Department of Physics, Gebze Institute of Technology, Cayirova Campus, PO Box 141, 41400 Gebze-Kocaeli, Turkey e-mail: zozturk@gyte.edu.tr H. Yu¨zer Z. Z. O¨ ztu¨rk TU¨ B_ITAK-Marmara Research Center, P.K.21, 41470 Gebze-Kocaeli, Turkey Introduction [19] synthesized Pd nanowires on Au sheet with AAO template using acidic electrolyte and applying constant voltage. Recently, Cheng et al. [20] prepared Pd nanowires on Ti substrate using AAO template by pulse electrodeposition for studying the electrocatalytic activity of Pd nanowires. Cherevko et al. [21] fabricated conoidal palladium nanowire and nanotube arrays by electrochemical deposition into the branched pores of an alumina template. Fig. 1 Schematic diagram of the preparation stages of the AAO template for Pd nanowire electrodeposition Mechanism of thin-ﬁlm metal deposition and structures of electrodeposits on metal substrates have been exten- sively studied using various electrochemical methods [22–24]. Despite the technological importance, the mech- anism of electrodeposition on a metallic substrate of metallic nanowires has not attracted much attention. Moreover, nucleation and growth of Pd nanowire array on a metal electrode by electrochemical deposition have not been studied yet in detail. More studies are needed to understand the complex nature and mechanism of the electrodeposition of the Pd nanowires on metal electrodes. Keeping such potential applications, a few studies on the nucleation and growth of metallic particles through elec- trodeposition processes were carried out during past dec- ades. In this paper, we focused on the fabrication of vertical Pd nanowire arrays in AAO template/Au/Ti structure by direct voltage electrodeposition. The fabrication process is similar to the study by Kartopu and his coworkers, but our electrolyte’s pH is neutral (7) and has fewer components. We reported new impressive results on the mechanism and kinetics of the initial stages of Pd electrodeposition in AAO template, nucleation and growth kinetics of Pd nanowires endowed with high crystallinity. Fig. 1 Schematic diagram of the preparation stages of the AAO template for Pd nanowire electrodeposition treated again in a 5 wt% phosphoric acid solution at 35C for 20 min to remove the AAO barrier layer. Schematic diagram of the preparation stages of the AAO template for Pd nanowire electrodeposition was given in Fig. 1. Electrodeposition was carried out in an aqueous solution containing Pd(NH3)4Cl2 with NH4Cl (pH value 7) at room temperature, using a three-electrode potentiostatic sys- tem(CHI 760C Electrochemical Workstation) with a satu- rated calomel electrode as a reference electrode and a graphite as a counter electrode. Pd was deposited using a potential of -1 V with 7,200 s deposition time. Introduction Metal nanowires are expected to play a major role in future nanotechnologies. Surface effect, small-size effect and even quantum effects are thus caused and severely affect the physical and chemical properties of these nanomaterials that differ signiﬁcantly from their bulk counterparts. Such unusual properties have generated interest in nanomaterials for potential applications in electronics, optics, magnetism, sensors, energy storage and catalysis [1–3]. Controllable growth of metallic nanowires has been a topic of contin- uing investigation, because of the need to obtain high- performance high-density nanoelectronic devices such as ﬁeld-effect transistors (FETs), chemical and biological sensors [4–7]. In order to prepare the metal nanowire arrays, it has been considered that the electrodeposition of the metal nanowires into a template is the simplest and most versatile approach since it does not utilize expensive and sophisticated lithographic processes for deﬁning nanomaterials [8–10]. The template method has been accomplished using a variety of templates, which are polycarbonate membranes, nanochannel alumina and anodized aluminum oxides (AAO). AAO template espe- cially is an ideal template to the synthesis of metal nano- wires as it has good mechanical strength and thermal stability with good controllability of nanowire dimensions [11–13]. Keywords Palladium Pd nanowires AAO template Electrodeposition Nucleation Nanomaterials To date, palladium is the most preferred active material for catalytic combustion, methanol oxidation, hydrogen sensors and hydrogen storage devices due to its high selectivity and afﬁnity to hydrogen. Hydrogen is adsorbed and dissociated spontaneously in palladium and its alloys [14, 15]. Therefore, Pd is a very special metal, but only a limited number of reports describe the fabrication of Pd nanowires [16–21]. Favier et al. [16] decorated the edge N. Tas¸altın S. O¨ ztu¨rk N. Kılınc¸ Z. Z. O¨ ztu¨rk (&) Department of Physics, Gebze Institute of Technology, Cayirova Campus, PO Box 141, 41400 Gebze-Kocaeli, Turkey e-mail: zozturk@gyte.edu.tr H. Yu¨zer Z. Z. O¨ ztu¨rk TU¨ B_ITAK-Marmara Research Center, P.K.21, 41470 Gebze-Kocaeli, Turkey 12 3 3 1138 Nanoscale Res Lett (2010) 5:1137–1143 steps on a graphite surface with Pd by electrodeposition using an electrolyte containing 2 mM Pd4? ions (in 0.1 M HCl or HClO4) and then obtained horizontal Pd nanowires. Then, Kim et al. [17] prepared Pd nanowire arrays with an aspect ratio of 10 using Si-based AAO template by pulsed- current electrodeposition and utilizing a commercial elec- trolyte [18]. Kartopu et al. Experimental Anodic aluminum oxide templates were fabricated through a two-step anodization of high-purity aluminum foils [25, 26]. The obtained AAO templates are prepared for Pd nanowire using electrodeposition method as follows: A thin gold ﬁlm was evaporated onto one surface of the AAO as-prepared template that serves as the working electrode attached with silver epoxy to Ti foil as substrate. The used Ti foil was polished and cleaned before. Then, the remaining aluminum of the AAO template, which was the opposite side of the Au ﬁlm, was etched in 5 wt% HgCl2 for complete removing. After that, the AAO template was Introduction Then, to obtain free-standing Pd nanowires, the AAO template was dissolved with a 5 wt% NaOH solution for 10 min and then carefully rinsed away with deionized water. X-ray dif- fraction (XRD, Cu Ka, k = 0.154 nm), scanning electron microscopy and energy-dispersive X-ray (SEM; EDX, Jeol JSM 6335) were used to study crystalline structures and morphologies of the fabricated Pd nanowires. 3HgCl2 þ 2Al ! 2AlCl3 þ 3Hg 3HgCl2 þ 2Al ! 2AlCl3 þ 3Hg The reaction products were cleaned from the remaining AAO template by rinsing with water. Hexagonal AAO barrier layers of all the AAO nanotubes were observed on the surface (Fig. 3a). The AAO barrier layers prevent the direct electrical contact between the AAO nanotubes and the Ti substrate, so that it is impossible to ﬁll the nanotubes with Pd by electrodeposition. For electrodeposition of Pd in AAO template to be possible, the AAO barrier layers were opened to a bottom electrode. To remove the barrier layer, AAO template was treated with 5 wt% phosphoric acid solutions at 35C for ca. 20 min. The SEM image of the obtained structure is given in Fig. 3b, which shows the surface of the AAO template with opened AAO barrier layer. Thus, the structure is ready for Pd electrodeposition. Nucleation and growth kinetics in the initial stages of Pd deposition on Au electrode in AAO template were studied using the current transient technique. For electrodeposition of Pd, although the acidic PdCl22HCl or K2PdCl4H2SO4 electrolytes, the alkaline Pd(NH3)4Cl2 or Pd(NH3)2(NO2)2 (P-salt type) electrolytes could be used, we worked with Pd(NH3)4Cl2 ? NH4Cl electrolyte as it is more chemically stable than the others. The mechanism of Pd electrode- position on Au electrode depends strongly on the Pd–Au The reaction products were cleaned from the remaining AAO template by rinsing with water. Hexagonal AAO barrier layers of all the AAO nanotubes were observed on the surface (Fig. 3a). The AAO barrier layers prevent the direct electrical contact between the AAO nanotubes and the Ti substrate, so that it is impossible to ﬁll the nanotubes with Pd by electrodeposition. For electrodeposition of Pd in AAO template to be possible, the AAO barrier layers were opened to a bottom electrode. To remove the barrier layer, AAO template was treated with 5 wt% phosphoric acid solutions at 35C for ca. 20 min. The SEM image of the obtained structure is given in Fig. 3b, which shows the surface of the AAO template with opened AAO barrier layer. Thus, the structure is ready for Pd electrodeposition. Nucleation and growth kinetics in the initial stages of Pd deposition on Au electrode in AAO template were studied using the current transient technique. Results and discussion The morphologies of the fabricated AAO template and Pd nanowire arrays were studied by SEM. Figure 2 shows 123 Nanoscale Res Lett (2010) 5:1137–1143 1139 SEM images of the fabricated AAO template after the second anodization process. The detailed fabrication of AAO template with two-step anodization was given pre- viously [25, 26]. The thickness and pore size of the AAO template can be controlled by changing the concentration of electrolytes, the time of anodic oxidation, time of pore widening and other conditions. Figure 2a shows top view of the AAO template, Fig. 2b shows a cross-sectional view of the AAO template and Fig. 2c shows a high magniﬁcation cross-sectional view of the AAO template. As clearly observed in Fig. 2, fabricated hexagonally straight AAO nanotubes were approximately 55 nm in diameter and 10 lm in length. The nanotubes exhibit almost perfect two-dimensional arrays with a hexagonal pattern. It shows that the formed nanotubes can be com- paratively homogeneous without distorting the structures, resulting in them being highly oriented in one direction with a high aspect ratio of approximately 182. From these results, fabricated AAO nanotube density is approxi- mately 2.59 9 1014 cm-2. The nanotube density (q) is calculated as 2/H3 Dint 2 9 1014 cm-2, where Dint is the interpore distance. ð1Þ 3HgCl2 þ 2Al ! 2AlCl3 þ 3Hg PdCl2 + 2NH4Cl ! PdCl4 ½ 2þ2NH3 þ 2Hþ ð2Þ ð2Þ The reaction starts after formation and charging of the double layer of hydronium ions (H??H2O = H3O?) and [PdCl4]2- ions on the Au electrode at the bottom of the AAO nanotubes. During the charging period, the current density decreases sharply with time. The [PdCl4]2- ions are reduced by accepting electrons from the electrode through hydrogen-bonded (H3O?) ions. The Pd?2 solution used for the electrodeposition was 0.0085 M, and when the Pd?2 solution ﬁlled a nanotube of the AAO template that has 5.94 9 106 nm3 volume, the number of the Pd?2 ions was approximately 30,258 in the mentioned AAO nanotube. To form a ﬁrst atomic Pd0 layer on the Au ﬁlm at the bottom of the AAO nanotube, the process required approximately 120,878 Pd?2 ions. Therefore, the required process was diffusion controlled, and double layers at the AAO nanotubes of the template could form very slowly for this electrodeposition process (the initial stage of current density is given in top right of Fig. 4). After forming the double layers, current density starts to increase continuously with nonlinear characteristics. Fig. 3 SEM images of the AAO template. a Before removing the alumina barrier layer. b After removing of the alumina barrier layer interaction. The kinetics and the mechanism of Pd elec- trodeposition and the involved phase formation phenomena can also be inﬂuenced signiﬁcantly by electronic properties of the Au substrate. Figure 4 shows the typical current transient for deposition of Pd. As shown in Fig. 4, the electrodeposition of Pd nano- wires is not a steady state process, and according to the Pd–Au interaction, the current–time transient shows a sharp drop in the initial current density due to the charging of the double layer, which was followed by a nonlinear increase in the current density with electrodeposition time. Such double-layer charging was also observed during electrodeposition of various metals [27–31]. But, on the contrary of previous studies, electrodeposition kinetic of Pd on Au electrode in AAO template, decreasing and then beginning to increase the current density, occurred in a very long time. Prior to the initiation of electrodeposition, palladium chloride forms [PdCl4]2-, palladium tetrachloro square-planar complex, with NH4Cl dissolved in electro- lyte as in the following reaction: The schematic view of growing of Pd in AAO template is given in Fig. 5. 3HgCl2 þ 2Al ! 2AlCl3 þ 3Hg For electrodeposition of Pd, although the acidic PdCl22HCl or K2PdCl4H2SO4 electrolytes, the alkaline Pd(NH3)4Cl2 or Pd(NH3)2(NO2)2 (P-salt type) electrolytes could be used, we worked with Pd(NH3)4Cl2 ? NH4Cl electrolyte as it is more chemically stable than the others. The mechanism of Pd electrode- position on Au electrode depends strongly on the Pd–Au As is seen in Fig. 3a, after dipping of the AAO template in HgCl2 solution, all of metallic Al was removed according to following cementation reaction (1). Fig. 2 SEM images of the fabricated AAO template. a Top view. b Cross-sectional view. c High-magniﬁcation cross-sectional view ages of the fabricated AAO template. a Top view. b Cross-sectional view. c High-magniﬁcation cross-sectional v Fig. 2 SEM images of the fabricated AAO template. a Top view. b Cross-sectional view. c High-magniﬁcation cr 12 3 Nanoscale Res Lett (2010) 5:1137–1143 1140 Fig. 3 SEM images of the AAO template. a Before removing the alumina barrier layer. b After removing of the alumina barrier layer Fig. 4 The current transient during the electrodeposition of the Pd in the AAO template Fig. 4 The current transient during the electrodeposition of the Pd in the AAO template PdCl2 + 2NH4Cl ! PdCl4 ½ 2þ2NH3 þ 2Hþ ð2Þ PdCl2 + 2NH4Cl ! PdCl4 ½ 2þ2NH3 þ 2Hþ ð2Þ The discharging of Pd0 to the Au elec- trode from the [PdCl4]2- solution can be expressed as follows: PdCl4 ½ 2þ2e ! Pd0 þ 4Cl ð3Þ ð3Þ Discharging of Pd atoms forms the nuclei on the Au electrodes at the bottom of the AAO nanotubes. Pd atoms were continuously deposited on the Au electrode during the electrodeposition. As the Au electrode is negative in charge, the hydronium ion was attracted toward the electrode, forming a positive layer on the deposited palladium particles followed by a negative layer of [PdCl4]2-. 123 123 1141 Nanoscale Res Lett (2010) 5:1137–1143 Fig. 5 The mechanism of Pd nucleation in AAO template Here, the value of the parabolic rate constant (kP) was obtained from the slope of current density–t1/2 transient. The electrodeposition current (I) usually obeys the following growth relation: where D is the diffusion coefﬁcient, c is the concentration of metallic ions in the solution, zF is the effective molar charge of the electrodepositing species and t is time. M and q are the molecular weight and density of the metallic species deposited, respectively. In order to calculate Pd nuclei density, the values of described parameters are used as follows; z = 2, F = 96,500 Coulombmol-1, D = 6.70 9 10-6 cm2s-1, M = 106.42 gmol-1, c = 2.087 9 10-6 molcm-3, and q = 12.023 gcm-3. Thus, for our nanodeposition, Pd nuclei density (N) is calculated as 3.55 9 108cm-2. This value is approximately 1.116 9 105 cm-2 for electrodeposition of Pd on the highly oriented pyrolitic graphite (HOPG) [30]. The values of the N estimated for electrodeposition of Pd on HOPG in experimental conditions other than the present situation have been reported to be 5.8 9 109 and 3.6 9 108 cm-2 [31, 32]. The differences in the values are due to applied Fig. 5 The mechanism of Pd nucleation in AAO template For understanding the growing mechanism of Pd nano- wires, the log (current density)-log (time1/2) transients need to be drawn (Fig. 6). It is clearly seen from Fig. 6 that current density in logarithmic scale is slowly decreased for a period and then decreased sharply to a minimum. After a steady state in this minimum for a period, the current density in logarithmic scale is increased. The increased part of log (current density)-log (time1/2) graph is shown in the right top of the ﬁgure. Conclusions Obtained vertically aligned Pd nanowires were approxi- mately 50 nm in diameters and 2.5 lm in lengths with aspect ratio of 46. For determination of optimum growing rate, consequently high grade of nanowire crystallinity, the initial stages of the electrodeposition of Pd nanowires on Au were studied using potentiostat. Nucleation rate and the number of atoms in the critical nucleus are determined from the analysis of current transients. The fabricated Pd nanowires are well arranged (Fig. 7), have high crystal- linity (Fig. 8) and also have less dislocations than fabri- cated known Pd nanowires due to slower nucleation rate. We believe that understanding the nucleation and growth mechanism in the electrodeposition process of Pd nano- wires gives rise to fabricate higher purity, higher crystal- linity, and lower dislocated Pd nanowires, suitable for potential nanotechnological applications especially in sensors. Figure 8a and b show a XRD spectrum and EDX spectra of the fabricated Pd nanowires, respectively. Fig. 8a con- ﬁrms that the Pd nanowires possessed a face-centered cubic (fcc) structure and were well crystallized. The strong dif- fraction peaks at the Bragg angles of 2h = 40.20, 46.78 and 68.10 correspond to the (1 1 1), (2 0 0) and (2 2 0) reﬂection lines ,respectively, of the fcc phase of palladium. cated known Pd nanowire We believe that understan mechanism in the electro wires gives rise to fabrica linity, and lower disloca potential nanotechnologic sensors. Acknowledgments This stud Technological Research Coun ‘‘Investigation and developme sors’’ and Project No. 106T546 Open Access This article is Creative Commons Attribution mits any noncommercial use, medium, provided the original References 1. A. Huczko, Appl. Phys. A 2. L. Vayssieres, Int. J Nanot 3. M.S. Dresselhaus, P. Avou Structure, Properties and A G. Dresselhaus, P.H. Avou 4. G. Zheng, W. Lu, S. Jin, C 5. E.S. Snow, F.K. Perkins Reinecke, Science 307, 19 6. M. Tian, J. Wang, Q. Zhan Chan, Nano Lett. 9, 3196 7. J. Wang, C. Shi, M. Tian, Mallouk, M.H.W. Chan, P 8. A.P. Li, F. Mu¨ller, A. Birn 11, 483 (1999) 9. H. Masuda, K. Fukuda, Sc Fig. 8 a XRD spectrum from the surface of the Pd nanowires. b EDX spectra from the surface of the Pd nanowires Fig. 8 a XRD spectrum from the surface of the Pd nanowires. PdCl2 + 2NH4Cl ! PdCl4 ½ 2þ2NH3 þ 2Hþ ð2Þ The slope of the log (current density)-log (time1/2) transient is approximately 0.13 Acm-2s-1/2, which indicates that the electrodepos- ition kinetic follows a parabolic relationship. This situation indicates an electrodeposition process of spontaneous nucleation and subsequent three-dimensional growth of formed nuclei. Therefore, the experimentally found out value of the number of the Pd nuclei density (N) can be calculated by using the following relation: Fig. 7 a SEM image of Pd nanowires after removing the AAO template. b High-magniﬁcation SEM image of Pd nanowires N ¼ q1=2 ZFpð2DcÞ3=2M1=2kP ð4Þ Fig. 6 Log (J)-log (t1/2) graph for Pd electrodeposition in AAO template. The increasing part of Log (J)-log (t1/2) graph is in the right top of the ﬁgure N ¼ q1=2 ZFpð2DcÞ3=2M1=2kP ð4Þ ð4Þ Fig. 6 Log (J)-log (t1/2) graph for Pd electrodeposition in AAO template. The increasing part of Log (J)-log (t1/2) graph is in the right top of the ﬁgure Fig. 7 a SEM image of Pd nanowires after removing the AAO template. b High-magniﬁcation SEM image of Pd nanowires 123 1142 Nanoscale Res Lett (2010) 5:1137–1143 potential, concentration of the Pd ions in electrodeposition solutions and pH of the electrodeposition solutions. The sharp (1 1 1) peak is good evidence of palladium nanowires with high crystallinity compared to the results of Kartopu et al. [19]. Furthermore, the EDX spectrum (Fig. 8b) shows strong Pd signals from the fabricated Pd nanowires and conﬁrms the electrodeposition of pure and high-crystalline Pd nanowires using dilute neutral electro- lyte as electrodeposition medium. Figure 7 shows the SEM images of Pd nanowires after dissolving the AAO template. It can be clearly observed from Fig. 7 that the Pd nanowires are highly ordered with uniform diameters approximately of 50 nm and lengths approximately 2.5 lm. The Pd nanowires are uniform and standing vertically to the electrode surface in a relatively large area, even though there are a few regions where the nanowires are absent. Therefore, the density of nanowires is almost close to the nanotube density of the fabricated AAO template. The Pd nanowires were not uniformly parallel to one another because of AAO-dissolving process in NaOH solution. Conclusions b EDX spectra from the surface of the Pd nanowires Acknowledgments This study was supported by the Scientiﬁc and Technological Research Council of Turkey under the project title ‘‘Investigation and development of nanotechnologic hydrogen sen- sors’’ and Project No. 106T546. 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https://openalex.org/W2774648133	https://www.beilstein-journals.org/bjoc/content/pdf/1860-5397-13-252.pdf	English	null	A novel synthetic approach to hydroimidazo[1,5-<i>b</i>]pyridazines by the recyclization of itaconimides and HPLC–HRMS monitoring of the reaction pathway	Beilstein journal of organic chemistry	2,017	cc-by	5,933	Abstract The novel cascade two-stage reaction between itaconimides and 1,2-diamino-4-phenylimidazole proceeds regio- and chemoselec- tively to form tetrahydroimidazo[1,5-b]pyridazines and includes nucleophilic C-addition by the activated C=C double bond and subsequent intramolecular recyclization of the intermediate with the amino group involved. Address: Michael Yu. Krysin* - kaf261@rambler.ru * Corresponding author A novel synthetic approach to hydroimidazo[1,5-b]pyridazines by the recyclization of itaconimides and HPLC–HRMS monitoring of the reaction pathway Dmitry Yu Vandyshev1 Khidmet S Shikhaliev1 Andrey Yu Potapov1 Full Research Paper Open Access Address: 1Faculty of Chemistry, Voronezh State University, Universitetskaya sq., 1, Voronezh 394018, Russian Federation and 2Faculty of Physics and Mathematics and Natural Sciences, RUDN University, Miklukho-Maklaya St., 6, Moscow 117198, Russian Federation Email: Michael Yu. Krysin* - kaf261@rambler.ru * Corresponding author Keywords: cascade reaction; diaminoimidazoles; HPLC–HRESIMS; imidazo[1,5-b]pyridazines; itaconimides Beilstein J. Org. Chem. 2017, 13, 2561–2568. doi:10.3762/bjoc.13.252 Received: 04 September 2017 Accepted: 14 November 2017 Published: 30 November 2017 Associate Editor: I. R. Baxendale © 2017 Vandyshev et al.; licensee Beilstein-Institut. License and terms: see end of document. Introduction of Hedgehog pathway dependent malignancies [7]. Imidazo[1,5-a]pyrimidines are inhibitors of the bone morphogenic protein [8], antitumor agents [9], and are stimula- tors of guanylate cyclase [10], whereas imidazo[1,5-c]pyrim- idines demonstrate anti-infectious effects in the treatment of brucellosis [11], etc. However, the chemistry, medicinal chem- istry and pharmacology of imidazo[1,5-b]pyridazines, with the imidazole ring connected through the N1–C5 bond of the latter, have been studied to a lesser extent. So, for imidazo[1,5-b]pyri- Among the numerous bicyclic fused imidazole derivatives, there is great interest in imidazo[1,5-annelated]diazines due to their diverse pharmacological actions and bioisosterism with imidazo[4,5-d]pyrimidines (purines). The presence of a bridge- head nitrogen atom is a common structural motif of these heterocyclic systems. For example, compounds with the imidazo[1,5-a]pyrazine structure show inhibitory activity against kinases BTK [1], MEK [2], ACK1 [3], mTORC1(2) [4], c-Src [5], growth factor IGF-1R [6] and act as the antagonists 2561 2561 Beilstein J. Org. Chem. 2017, 13, 2561–2568. tures in drug design [22], we decided to search for new reagents for the synthesis of their imidazo[1,5-b]-annelated analogues. dazines, inhibitory activity was found against the viruses of human immunodeficiency HIV-1 [12], influenza [13], and hepatitis C [14]. They also act as stimulators of guanylate cyclase [10], inhibitors of phosphodiesterase 10A [15], protease-activated receptor 1 [16], PIM-1/2 kinase [17], antago- nists of the corticotropin releasing factor [18,19], vanilloid-1 re- ceptor [20], and modulators for ligand binding to GABAA re- ceptor [21]. As pyridazines are considered as privileged struc- There are two general approaches to the construction of an imidazo[1,5-b]pyridazine scaffold: the annulation of the imida- zole ring to the b-bond of the pyridazine (route A, Scheme 1) and the heterocyclization of 1-aminoimidazoles to build the pyridazine core (route B, Scheme 2 and Scheme 3). Scheme 1: Intramolecular cyclization of 3-(aminomethyl)pyridazines and related compounds (route A). Conditions: i) R2COCl/Et3N [12], R2COOH/ DCC [13,23], (R2CO)2O/Et3N [19]; ii) synthetic equivalent of C1-electrophile: R2COOH/T3P® [15], BrCN (R2 = NH2) [16], ArNCS/DCC (R2 = NHAr) [20]. Scheme 1: Intramolecular cyclization of 3-(aminomethyl)pyridazines and related compounds (route A). Conditions: i) R2COCl/Et3N [12], R2COOH/ DCC [13,23], (R2CO)2O/Et3N [19]; ii) synthetic equivalent of C1-electrophile: R2COOH/T3P® [15], BrCN (R2 = NH2) [16], ArNCS/DCC (R2 = NHAr) [20]. Scheme 2: Heterocyclization of 1-aminoimidazoles with 1,3-dicarbonyl or α,β-unsaturated carbonyl compounds (route B). Introduction Further, by careful selectio tants functional diversification of the target imid over virtually any carbon atom of the heterocy achievable in addition to hydrogenated imida dazines [34,35]. Itaconimides, in contrast to itaconic aci monoamides or anhydride [36,37], scarcely hav in the synthesis of heterocyclic compounds. The exocyclic activated C=C double bond allows it react with heteroaromatic dinucleophiles as d reagents with the possibility of recyclization. group reported the first example of this recy N-arylitaconimides 1 and 1,2-diaminobenzim 1,3-N,N-dinucleophile, leading to tetrahydro a]benzimidazoles 3 (Scheme 4) [38]. In continuation of our studies on the synthe annelated heterocycles we herein report the resul tigations on the reactions between N-arylitaconi diamino-4-phenylimidazole as a heterocyclic C nucleophile. Results and Discussion Our earlier studies and literature information ha Scheme 4: Recyclization of N-arylitaconimides 1 with 1,2-diamino- benzimidazole (2). Following route A (Scheme 1), the aminomethylpyridazine de- rivatives are either pre-acylated and subsequently cyclized with phosphorus oxychloride [12,13,19,23], or the formation of imidazo[1,5-b]pyridazines occurs as a result of their direct heterocyclization involving various single-carbon electrophilic reagents [15,16,20]. A similar one-pot aza-Staudinger reaction of 3-(azidomethyl)pyridazines with isothiocyanates in the pres- ence of PMe3 also results in imidazopyridazines [17]. Following route A (Scheme 1), the aminomethylpyridazine de- rivatives are either pre-acylated and subsequently cyclized with phosphorus oxychloride [12,13,19,23], or the formation of imidazo[1,5-b]pyridazines occurs as a result of their direct heterocyclization involving various single-carbon electrophilic reagents [15,16,20]. A similar one-pot aza-Staudinger reaction of 3-(azidomethyl)pyridazines with isothiocyanates in the pres- ence of PMe3 also results in imidazopyridazines [17]. Scheme 4: Recyclization of N-arylitaconimides 1 with 1,2-diamino- benzimidazole (2). In the synthesis of imidazo[1,5-b]pyridazines along route B, 1-aminoimidazoles with no substituents at the C-5 atom of the original heterocycle and 1,3-dielectrophilic reagents are used, e.g., 1,3-diketones [24-26], β-ketoesters [24], α,β-unsaturated ketones [27,28], including those obtained in situ [29,30] or con- taining good leaving groups [31,32] (Scheme 2). diaminoimidazole with 1,3-dielectrophilic reagents. Therefore, the heterocyclization of N-arylitaconimides 1a–g and diaminoimidazole 4 was carried out in refluxing iPrOH in the presence of catalytic amounts of AcOH. In all reactions only one compound was isolated in good yields (Table 1). Attempts to replace the alcoholic solvent with dioxane or DMF resulted in decreased yields of the target products. In the reactions of 1-aminoimidazoles with cyclic 1,3-dielec- trophilic reagents (3-formylchromones [33], Meldrum’s acid derivatives [34], and N-arylmaleimides [35]), transformat- ion of the structure of the latter ones is often observed (Scheme 3). Introduction Table 1: Yields of the interaction products 9 of N-arylitaconimides 1a–g and diaminoimidazole 4. entry itaconimide Ar product yield (%) 1 1a 4-MeC6H4 9a 62 2 1b 4-EtC6H4 9b 60 3 1c 3-ClC6H4 9c 70 4 1d 4-ClC6H4 9d 72 5 1e 3,4-Me2C6H3 9e 65 6 1f 3,5-Me2C6H3 9f 67 7 1g 3,4-Cl2C6H3 9g 66 Table 1: Yields of the interaction products 9 of N-arylitaconimides 1a–g and diaminoimidazole 4. Table 1: Yields of the interaction products 9 of N-arylitaconimides 1a–g and diaminoimidazole 4. Among these methods the heterocyclization (method B) of 1-aminoimidazoles seems more attractive due to their better accessibility when compared to the corresponding pyridazines required in route A. Further, by careful selection of the reac- tants functional diversification of the target imidazopyridazines over virtually any carbon atom of the heterocyclic system is achievable in addition to hydrogenated imidazo[1,5-b]pyri- dazines [34,35]. Itaconimides, in contrast to itaconic acid, its esters, monoamides or anhydride [36,37], scarcely have been studied in the synthesis of heterocyclic compounds. The presence of the exocyclic activated C=C double bond allows itaconimides to react with heteroaromatic dinucleophiles as dielectrophilic reagents with the possibility of recyclization. Recently, our group reported the first example of this recyclization with N-arylitaconimides 1 and 1,2-diaminobenzimidazole (2) as 1,3-N,N-dinucleophile, leading to tetrahydropyrimido[1,2- a]benzimidazoles 3 (Scheme 4) [38]. The problem of chemoselectivity and regioselectivity of the reactions under study is associated with the polynucleophilicity of 1,2-diamino-4-phenylimidazole (4), whose structure contains several dinucleophilic centers, namely: 1,3-C,N (C5-N1-NH2), 1,3-N,N (HN3-C2=NH in the other tautomeric form 4-I), 1,4-N,N (NH2-N1-C2-NH2) as well as with the polyelec- trophilicity of itaconimides 1 due to the presence of three elec- trophilic C atoms: the terminal atom of the exocyclic activated multiple bond and two atoms of the imide group. Due to this, at the first step of the reaction between diaminoimidazole and itaconimides, four primary adducts 5–8 could result (Scheme 5) based on the well-known fact that the heterocyclization reac- tions of α,β-unsaturated carbonyl compounds with dinucleo- philic reagents typically begin with addition like Michael’s reaction, including heterocyclic dinucleophiles [39-42]. Mecha- nistically, further intramolecular recyclization of the succin- imide fragments in intermediates 5–8 could proceed by any of the two imide C atoms (Scheme 5). In continuation of our studies on the synthesis of azolo- annelated heterocycles we herein report the results of our inves- tigations on the reactions between N-arylitaconimides and 1,2- diamino-4-phenylimidazole as a heterocyclic C,N/N,N poly- nucleophile. Introduction Conditions: i) R1 = NH2, NHAlk, R2 = Ph, R3,4 = Alk, Ar, solvent-free [24], AcOH [25,26], R3 = Ph, R4 = OEt, solvent-free [24]; ii) R1 = NH2, R2 = Ph, R3 = H, Me, R4 = Ar, ArCHO/MeOH/DMF [29] or R4 = H, HC(OEt)3/iPrOH [30]; iii) R1 = NH2, R2 = Ar, R3,4 = Alk, Ar, MeOH/N-methylmorpholine or DMF or AcOH [27,28], R3 = Ar, R4 = COOH, DMF [31], R3 = Ar, R4 = NMe2, AcOH/DMF [32]. Scheme 2: Heterocyclization of 1-aminoimidazoles with 1,3-dicarbonyl or α,β-unsaturated carbonyl compounds (route B). Conditions: i) R1 = NH2, NHAlk, R2 = Ph, R3,4 = Alk, Ar, solvent-free [24], AcOH [25,26], R3 = Ph, R4 = OEt, solvent-free [24]; ii) R1 = NH2, R2 = Ph, R3 = H, Me, R4 = Ar, ArCHO/MeOH/DMF [29] or R4 = H, HC(OEt)3/iPrOH [30]; iii) R1 = NH2, R2 = Ar, R3,4 = Alk, Ar, MeOH/N-methylmorpholine or DMF or AcOH [27,28], R3 = Ar, R4 = COOH, DMF [31], R3 = Ar, R4 = NMe2, AcOH/DMF [32]. Scheme 3: Heterocyclization of 1-aminoimidazoles with structural transformation of dielectrophilic reagents (route B). Conditions: i) R1 = NH2, SH, R2 = H, Me, MeO, F, Me3SiCl/DMF [33]; ii) R1 = NH2, MeOH/DMF [34]; iii) R1 = NH2, AcOH/iPrOH [35]. Scheme 3: Heterocyclization of 1-aminoimidazoles with structural transformation of dielectrophilic reagents (route B). Conditions: i) R1 = NH2, SH, R2 = H, Me, MeO, F, Me3SiCl/DMF [33]; ii) R1 = NH2, MeOH/DMF [34]; iii) R1 = NH2, AcOH/iPrOH [35]. 2562 Beilstein J. Org. Chem. 2017, 13, 2561–2568. Following route A (Scheme 1), the aminomethyl rivatives are either pre-acylated and subsequently phosphorus oxychloride [12,13,19,23], or the imidazo[1,5-b]pyridazines occurs as a result heterocyclization involving various single-carbo reagents [15,16,20]. A similar one-pot aza-Staud of 3-(azidomethyl)pyridazines with isothiocyana ence of PMe3 also results in imidazopyridazines In the synthesis of imidazo[1,5-b]pyridazines 1-aminoimidazoles with no substituents at the C original heterocycle and 1,3-dielectrophilic rea e.g., 1,3-diketones [24-26], β-ketoesters [24], α ketones [27,28], including those obtained in situ taining good leaving groups [31,32] (Scheme 2). In the reactions of 1-aminoimidazoles with cyc trophilic reagents (3-formylchromones [33 acid derivatives [34], and N-arylmaleimides [35] ion of the structure of the latter ones is of (Scheme 3). Among these methods the heterocyclization ( 1-aminoimidazoles seems more attractive due accessibility when compared to the correspondi required in route A. Results and Discussion Our earlier studies and literature information have shown that acetic acid significantly accelerates the reactions of 1,2- 2563 Beilstein J. Org. Chem. 2017, 13, 2561–2568. Scheme 5: Possible synthetic routes of the interaction of itaconimides 1 with diaminoimidazole 4. Scheme 5: Possible synthetic routes of the interaction of itaconimides 1 with diaminoimidazole 4. an amino group at the second position of the cycle [43]. The heterocyclization of intermediate 6 involving the C5 atom of the imidazole is improbable (not shown in the Scheme 5). Thus, for products 5 of the reaction of diaminoimidazole 4 as a 1,3-C,N-dinucleophile in the routes A and B, imidazo[1,5-b]pyridazines 9 and imidazo[1,5-b][1,2]diazepines 10, respectively, can be obtained. As a result of the initial addi- tion of diaminoimidazole 4 as a 1,4-N,N-dinucleophile, inter- mediates 6 and 7 could be formed, whose heterocyclization with a second amino group could lead to imidazotriazepines 11 and 13 or imidazotriazocines 12 and 14. The capability of diaminoimidazoles in their tautomeric form 4-I to react as 1,3-N,N-dinucleophiles [38] determines the possible formation of adducts 8, whose recyclization could give imidazopyrimi- dines 15 or imidazodiazepines 16. The amine–imine tautomeric equilibrium is characteristic of aminoazole systems containing The key criterion for the choice of the structure of the com- pounds obtained is the presence of the signals of four protons associated with nitrogen atoms in their 1H NMR spectra: the broad singlet of the NH2 group in the region of 5.6–5.7 ppm and those of the two amide NH groups in the region of 10.0–11.5 ppm. This set of signals unambiguously excludes the possible structures of both intermediates 6–8 and their recy- clization products 11–16, as these molecules contain only three hydrogen atoms in their nitrogen-containing functional groups. 2564 Beilstein J. Org. Chem. 2017, 13, 2561–2568. The final choice in favor of the formation of imidazo- pyridazines 9a–g is based on the correlations found in the 1H,13C-HMBC NMR spectrum of compound 9d (Scheme 6). To confirm the presence of the last six-membered pyridazine ring in the structure and, accordingly, the exocyclic acetanilide residue, it is first of all necessary to make a strict assignment of the carbonyl carbon atom signals at around 170 ppm. ever, the corresponding cross peaks were not detected, due to the spatial remoteness of these hydrogen atoms. Results and Discussion Moreover, for this atom there are also two correlations with one of the dia- stereotopic protons of the exocyclic methylene group, whose signal (~2.60 ppm) overlaps with the residual proton signals of DMSO-d6, and with the proton of the methine group (2.89 ppm), which is partially overlapped by the signal of water present in the solvent. As no cross-peaks with protons of another methylene group are observed due to a significant num- ber of bonds between them, the acetanilide moiety must be in the exo-position to the pyridazine ring. One of the remaining protons (3.32 ppm) correlates with the carbonyl C atom (170.23 ppm) of the lactam fragment. In the alternative struc- ture of imidazodiazepine 10d, the amide carbon atom would correlate with the protons of both methylene as well as methine groups (Scheme 6). In the 1H NMR spectra of imidazopyridazines 9a–g, there are two N–H singlets: a narrow one at 10.19 ppm and a broadened at 11.35 ppm. The type of signals allows making an assumption that the latter refers to a cyclic lactam fragment whose hydro- gen atoms are sufficiently mobile. However, only for the narrow singlet there are cross peaks with a carbonyl C atom at 169.58 ppm and the carbon atom of the benzene ring (120.55 ppm) in the 1H,13C-HMBC spectrum. For the last aro- matic carbon atom, there is also a correlation with the proton of the ring (7.59 ppm). Therefore, the above mentioned C-carbon- yl is highly likely to belong to the amide group. Moreover, for this atom there are also two correlations with one of the dia- stereotopic protons of the exocyclic methylene group, whose signal (~2.60 ppm) overlaps with the residual proton signals of DMSO-d6, and with the proton of the methine group (2.89 ppm), which is partially overlapped by the signal of water present in the solvent. As no cross-peaks with protons of another methylene group are observed due to a significant num- ber of bonds between them, the acetanilide moiety must be in the exo-position to the pyridazine ring. One of the remaining protons (3.32 ppm) correlates with the carbonyl C atom (170.23 ppm) of the lactam fragment. In the alternative struc- ture of imidazodiazepine 10d, the amide carbon atom would correlate with the protons of both methylene as well as methine groups (Scheme 6). Results and Discussion The registration of the 1H NMR spectrum of imidazopyridazine 9d in the presence of trace amounts of CF3COOH allowed for a better assignment of the aliphatic protons due to a change in the position and intensity of signals of those protons associated with heteroatoms capable of protonation. This also included protons of water present in the DMSO-d6. Thus, doublets of doublets were observed for the diastereotopic protons of the exocyclic methylene group at 2.64 ppm (2J = 15.9 Hz, 3J = 6.7 Hz) and 2.87 ppm (2J = 15.9 Hz, 3J = 5.9 Hz) in addi- tion the protons of the endocyclic methylene group at 3.09 ppm (2J = 15.6 Hz, 3J = 10.6 Hz) and 3.32 ppm. (2J = 15.6 Hz, 3J = 5.5 Hz). The signal of the methine proton was a multiplet (3.17–3.25 ppm). The addition of trifluoroacetic acid did not have a significant effect on the position of the signals of these protons. Scheme 6: 1H,13C-HMBC correlations: the most significant correla- tions for imidazopyridazine 9d and possible for imidazodiazepine 10d. Scheme 6: 1H,13C-HMBC correlations: the most significant correla- tions for imidazopyridazine 9d and possible for imidazodiazepine 10d. Thus, the reaction of itaconimides 1 with diaminoimidazole 4 is a regioselective and chemoselective cascade process involving an initial C-addition of diaminoimidazole as a 1,3-C,N- dinucleophile to the activated C=C double bond to form inter- mediate 5 followed by recyclization involving the N1-amino group which leads to the formation of imidazo[1,5-b]pyri- dazines 9. In the 1H NMR spectra of imidazopyridazines 9a–g, there are two N–H singlets: a narrow one at 10.19 ppm and a broadened at 11.35 ppm. The type of signals allows making an assumption that the latter refers to a cyclic lactam fragment whose hydro- gen atoms are sufficiently mobile. However, only for the narrow singlet there are cross peaks with a carbonyl C atom at 169.58 ppm and the carbon atom of the benzene ring (120.55 ppm) in the 1H,13C-HMBC spectrum. For the last aro- matic carbon atom, there is also a correlation with the proton of the ring (7.59 ppm). Therefore, the above mentioned C-carbon- yl is highly likely to belong to the amide group. Results and Discussion Monitoring of the liquid phase composition of the reaction mix- ture by HPLC–HRMS showed that during the reaction of itaconimide 1d with diaminoimidazole 4, four compounds result with the integer mass of the protonated molecular ion (m/z 396 [M + H]+), which corresponds to the possible products of the reagent interaction (Table 2). However, it is still impossible to give a full assessment of the probable routes of the cascade recyclization process, because ions of the protonable substances are only fixed in the given ESI–MS conditions, and precipitation of the product is ob- served as the reaction proceeds. The latter causes a decreased content of the imidazopyridazine 9d in the liquid phase is ob- served, whose peak is identified by the retention time (4.13 min) determined for the pure substance. The long reten- tion time (5.6 min) and the insignificant content (less than 1%) of the initial itaconimide 1d found under ESI conditions in the reaction mixture are due to its extremely low proton affinity. Allowing for the formal structural similarity of intermediates 5–8d, we assume that one of the chromatographic peaks with the retention time of 3.6 or 3.8 min corresponds to the interme- diate diaminoimidazole 5d, and the second one corresponds to one of succinimides 6–8d. The accumulation of the compound exhibiting a retention time of 5.3 min occurs about 30 min after A possible confirmation of the formation of imidazopyridazine 9d would be the correlation between protons of the exo- and endocyclic methylene groups in the NOESY experiment. How- 2565 Beilstein J. Org. Chem. 2017, 13, 2561–2568. Table 2: Results of HPLC–HRESIMS monitoring of the reaction mixture composition in the synthesis of imidazopyridazine 9d. Table 2: Results of HPLC HRESIMS monitoring of the reaction mixture composition in the synthesis of imidazopyridazine 9d. ORCID® iDs Dmitry Yu. Vandyshev - https://orcid.org/0000-0001-8606-458X Khidmet S. Shikhaliev - https://orcid.org/0000-0002-6576-0305 Andrey Yu. Potapov - https://orcid.org/0000-0001-8084-530X Michael Yu. Krysin - https://orcid.org/0000-0002-4336-8935 Fedor I. Zubkov - https://orcid.org/0000-0002-0289-0831 Lyudmila V. Sapronova - https://orcid.org/0000-0001-5811-6709 Dmitry Yu. Vandyshev - https://orcid.org/0000-0001-8606-458X Khidmet S. Shikhaliev - https://orcid.org/0000-0002-6576-0305 Andrey Yu. Potapov - https://orcid.org/0000-0001-8084-530X Michael Yu. Krysin - https://orcid.org/0000-0002-4336-8935 Fedor I. Zubkov - https://orcid.org/0000-0002-0289-0831 Lyudmila V. Sapronova - https://orcid.org/0000-0001-5811-6709 Dmitry Yu. Vandyshev - https://orcid.org/0000-0001-8606-458X Khidmet S. Shikhaliev - https://orcid.org/0000-0002-6576-0305 Andrey Yu. Potapov - https://orcid.org/0000-0001-8084-530X Michael Yu. Krysin - https://orcid.org/0000-0002-4336-8935 Fedor I. Zubkov - https://orcid.org/0000-0002-0289-0831 Lyudmila V. Sapronova - https://orcid.org/0000-0001-5811-6709 References 2016, 7, 198–203. doi:10.1021/acsmedchemlett.5b00463 2. 2. Robarge, K. D.; Lee, W.; Eigenbrot, C.; Ultsch, M.; Wiesmann, C.; Heald, R.; Price, S.; Hewitt, J.; Jackson, P.; Savy, P.; Burton, B.; Choo, E. D.; Pang, J.; Boggs, J.; Yang, A.; Yang, X.; Baumgardner, M. Bioorg. Med. Chem. Lett. 2014, 24, 4714–4723. doi:10.1016/j.bmcl.2014.08.008 Supporting Information Supporting Information File 1 Experimental procedures, characterization data, copies of 1H, 13C spectra of the products and results of HPLC–HRESIMS monitoring of the reaction mixture composition. [http://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-13-252-S1.pdf] 3. Jin, M.; Wang, J.; Kleinberg, A.; Kadalbajoo, M.; Siu, K. W.; Cooke, A.; Bittner, M. A.; Yao, Y.; Thelemann, A.; Ji, O.; Bhagwat, S.; Mulvihill, K. M.; Rechka, J. A.; Pachter, J. A.; Crew, A. P.; Epstein, D.; Mulvihill, M. J. Bioorg. Med. Chem. Lett. 2013, 23, 979–984. doi:10.1016/j.bmcl.2012.12.042 3. Jin, M.; Wang, J.; Kleinberg, A.; Kadalbajoo, M.; Siu, K. W.; Cooke, A.; Bittner, M. A.; Yao, Y.; Thelemann, A.; Ji, O.; Bhagwat, S.; Mulvihill, K. M.; Rechka, J. A.; Pachter, J. A.; Crew, A. P.; Epstein, D.; Mulvihill, M. J. Bioorg. Med. Chem. Lett. 2013, 23, 979–984. doi:10.1016/j.bmcl.2012.12.042 Acknowledgements the beginning of the reaction. In our opinion, this minor prod- uct is either imidazodiazepine 10d or one of the possible prod- ucts 11–16d of recyclization of intermediates 6–8d due to their more complex structure compared with the structure of the latter ones, and, correspondingly, the lower chromatographic mobility. However, the formation of heterocyclic systems in- cluding 7- and 8-membered rings is unlikely, because of the spatial remoteness of the corresponding reaction centers in the recyclization process. Nevertheless, the results of the HPLC–HRESIMS monitoring of the reaction confirm its selec- tivity. the beginning of the reaction. In our opinion, this minor prod- uct is either imidazodiazepine 10d or one of the possible prod- ucts 11–16d of recyclization of intermediates 6–8d due to their more complex structure compared with the structure of the latter ones, and, correspondingly, the lower chromatographic mobility. However, the formation of heterocyclic systems in- cluding 7- and 8-membered rings is unlikely, because of the spatial remoteness of the corresponding reaction centers in the recyclization process. Nevertheless, the results of the HPLC–HRESIMS monitoring of the reaction confirm its selec- tivity. This work was supported by the Ministry of Education and Science of the Russian Federation (Agreement number 02.a03.21.0008). Results and Discussion entry compound [M + H]+ calcd m/z [M + H]+ found m/z tRa, min composition of the reaction mixture, % (time after reaction start) 10 min 11 min 16 min 30 min 60 min 1 1d 222.0317 222.0314 5.6 0.7 0.4 – – – 2 4 175.0979 175.0977 1.5 81.2 83.3 81.2 77.6 79.9 3 5–8db 396.1223 396.1225 3.6 2.0 1.9 1.7 2.1 2.2 4 5–8db 396.1223 396.1225 3.8 5.3 5.1 7.8 10.5 11.2 5 9dc 396.1223 396.1224 4.2 10.8 8.8 8.6 7.4 3.5 6 10dd 396.1223 396.1224 5.3 – 0.5 0.7 2.4 3.2 aRetention time (tR), average value; bone of possible intermediates 5–8d; cfor isolated compound 9d, the retention time is 4.13 min; dimidazodiazepine 10d or one of the possible products of recyclization of intermediates 6–8d. aRetention time (tR), average value; bone of possible intermediates 5–8d; cfor isolated compound 9d, the retention time is 4.13 min; dimidazodiazepine 10d or one of the possible products of recyclization of intermediates 6–8d. 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https://openalex.org/W2768920098	https://europepmc.org/articles/pmc5989179?pdf=render	English	null	Is malaria over-diagnosed? A world malaria day 2017 experience by excellence and friends management care centre (EFMC) and partners, Abuja Nigeria	The Pan African medical journal	2,017	cc-by	3,288	Pan African Medical Journal – ISSN: 1937- 8688 (www.panafrican-med-journal.com) Published in partnership with the African Field Epidemiology Network (AFENET). (www.afenet.net) Received: 12/05/2017 - Accepted: 20/08/2017 - Published: 28/11/2017 Received: 12/05/2017 - Accepted: 20/08/2017 - Published: 28/11/2017 Open Access Abstract Malaria remains a major cause of mortality across the world, but particularly in sub-Saharan Africa. WHO-sponsored World Malaria Day activity has helped to improve education and has contributed to a reduction in mortality globally in the past decade. However, much needs to be done still in Africa. We report on a World Malaria Day scheme in three primary Healthcare Facilities in and around the Abuja Federal Capital Territory in Nigeria in 2017. Activity included educational talks to pregnant women and nursing mothers of young children, with malarial testing, distribution of free mosquito nets and also medical treatment if needed. We found a large clinical over-diagnosis of malaria with simple fevers of any cause being reported as malaria. None of these cases were found to be due to malaria on formal malarial testing. We conclude that efforts should continue into education and prevention of malaria with insecticide-impregnated mosquito nets a key factor. However, over-diagnosis of malaria and the use of unnecessary antimalarial treatment may lead to parasite resistance to antimalarial treatment, morbidity from drug side-effects and potential mortality from not receiving the right treatment for other febrile illnesses. We recommend that malarial testing, particularly with simple blood film microscopy is implemented more widely across Africa, as it is simple to perform and allows effective management plans to be drawn up for individual patients. Obinna Ositadimma Oleribe1,2,3,&, Princess Osita-Oleribe1,2, Ekei Ekom1, Oriaku Ofem1, Chidi Igwesi1, Obison Guy Chigozie1, Munaonyeso Ekweghariri1, Grace Iyalla1, Simon David Taylor-Robinson3,4 Obinna Ositadimma Oleribe1,2,3,&, Princess Osita-Oleribe1,2, Ekei Ekom1, Oriaku Ofem1, Chidi Igwesi1, Obison Guy Chigozie1, Munaonyeso Ekweghariri1, Grace Iyalla1, Simon David Taylor-Robinson3,4 1Excellence and Friends Management Care Centre (EFMC), N° 8 Excellence and Friends Street, Dutse, Abuja Nigeria, 2Centre for Family Health Initiative (CFHI), Plot 508 Excellence and Friends Road, Kubwa, Abuja Nigeria, 3Royal College of Physicians of London, 11 St Andrews Place, Regent’s Park, London NW1 4LE, 4Faculty of Medicine, Imperial College London, St Mary’s Hospital Campus, Norfolk Place, London W2 1PG, United Kingdom &Corresponding author: Obinna Ositadimma Oleribe, Excellence and Friends Management Care Centre (EFMC), No 8 Excellence and Friends Street, Dutse, Kubwa Abuja Nigeria &Corresponding author: Obinna Ositadimma Oleribe, Excellence and Friends Management Care Centre (EFMC), No 8 Excellence and Friends Street, Dutse Kubwa Abuja Nigeria Key words: EFMC, Malaria, over-diagnosis, rapid diagnostic test, Nigeria Received: 12/05/2017 - Accepted: 20/08/2017 - Published: 28/11/2017 Introduction Malaria is a preventable and curable/treatable illness transmitted by infected female Anopheles mosquitoes. According to the World Health Organization (WHO), in 2015, 91 countries and areas had ongoing malaria transmission [1]. However, increased efforts are dramatically reducing the malaria burden in many places. Between 2010 and 2015, malaria incidence among populations at risk fell by 21% globally [1]. In that same period, malaria mortality rates among populations at risk fell by 29% globally among all age groups and by 35% among children under 5. However, the WHO African Region carries a disproportionately high share of the global malaria burden and was home to 90% of malaria cases and 92% of malaria deaths in 2015 [1]. In sub-Saharan Africa, the high malaria burden is coupled with high rates of poverty and very low access to adequate health care. Efforts aimed at preventing the spread of malaria are very important to reduce the malaria burden. This informed the theme for 2017 World Malaria Day (WMD) with "a push for prevention" given that malarial drug resistance is increasing, the prevention of malaria is pertinent to reducing deaths and protecting vulnerable people, such as nursing mothers and young children. In aligning with this year's prevention drive, Excellence and Friends Management Care Centre (EFMC), a NGO based in Abuja, Nigeria, conducted health talks, free malaria testing, distribution of insecticide-treated mosquito nets and provided free drugs to women and children at three primary health care centers (PHC), in and around the Abuja Nigerian Federal Capital Territory. This report is a description of the events at these three PHCs. Malaria diagnosis and ITN distribution: Free malaria tests were conducted using rapid test kits (One Step Malaria P.f HRH -II Antigen Rapid TEST One Step Malaria P.f HRH-II Antigen Rapid TEST [2] for 79 (67.5%) women and children who had a febrile illness. Similarly, 78 (66.7%) insecticide-treated mosquito nets were distributed to qualified pregnant women and nursing mothers Figure 4, Figure 5. Outcome of malaria testing: None of the women and children tested was identified to have malaria from the test. This is despite the fact that they had clinical symptoms similar to malaria like fever. Responses from beneficiaries: Beneficiaries were excited about the services and knowledge acquired on how to prevent malaria. The beneficiaries also took turns to show their appreciation in their local languages from several ethnic groups of Nigeria. Methods Excellence and Friends Management Care Centre (EFMC) partnered with the Center for Family Health Initiative (CFHI) in Abuja in the implementation of free medical services to pregnant and nursing women, and children under the age of 5 yrs living in and around the Nigerian capital city. Mosquito nets and rapid diagnostic kits were provide by EFMC, CFHI and the Nigerian National Malaria Elimination Program (NMEP). Following several preparatory meetings, three sites-Kagini, Gwagwa and Idu-Karmo Primary Health Care Centers were purposefully selected (Figure 1). Criteria for selection included location, population covered and antenatal/well baby clinics on World Malaria Day (WMD). Selected sites were informed of their choice as well as the plans to provide free medical services with ITN distribution on WMD. Following consent, EFMC and CFHI designated staff members were divided into three teams led by Program Managers or clinical associates. Each team of 10 persons had testers, counsellors, doctors, community health workers and other support staff. Activity at each center was to start as early as 0800 hours and continued until the antenatal or well-baby clinic was over. EFMC/CFHI staff worked with staff of the facility to ensure their full involvement and proper documentation of the exercise Figure 2. Introduction In the words of some of the beneficiaries: "We are glad for the knowledge acquired today, the free test and free net. Thank God someone remembered us in recession" LE "What happened today is a good thing. I have leant how I can protect my family" MU "We give God thanks for the good works of EFMC" RA. Discussion World Malaria Days are dedicated days designed to help improve awareness of the impact of malaria, prevention strategies and treatment options available in the communities. Our activity showed that not all malaria-like fevers in malaria-endemic regions like Nigeria are as a result of malaria [3, 4]. There are several publications on the dangers of presumptive or clinical diagnosis of malaria. There are several publications on the dangers of presumptive or clinical diagnosis of malaria, resulting in false positive diagnosis and unnecessary treatment. In a study in Lagos, Nigeria, over-diagnosis and over-treatment of malaria was seen, of children treated with antimalarials were confirmed to be parasite positive as only 251 of 853 (29.4%) of children treated with antimalarials were confirmed to be parasite positive [3]. In a similar study on malaria burden in the neighbouring Republic of Niger, the number of cases of presumed malaria reported in health centers were said to be largely overestimated as a result of inadequacies in the clinical case ascertainment of the malaria and of disease risk, as patients with simple febrile illness were often wrongly classified as malaria cases [4]. In our own field work, although the subjects had clinical pyrexia, malaria tests were negative, again confirming that many fevers in Nigeria may not be as a result of malaria. Studies from Tanzania [5], Namibia [6] and several other nations have documented similar findings which all shown that diagnosis of Pan African Medical Journal. 2017; 28:273 doi:10.11604/pamj.2017.28.273.12732 Pan African Medical Journal. 2017; 28:273 doi:10.11604/pamj.2017.28.273.12732 This article is available online at: http://www.panafrican-med-journal.com/content/article/28/273/full/ © Obinna Ositadimma Oleribe et al. The Pan African Medical Journal - ISSN 1937-8688. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Health talks: The program began with a health talk upon the arrival of the project team at the designated facility. In most facilities, the beneficiaries, made up of women and children, were already seated in anticipation of the proposed medical services. Health talks on healthy living, malaria symptoms, preventive strategies including use of ITNs, indoor residual sprays and malaria chemoprophylaxis for the vulnerable population were undertaken; and management of fevers were presented to the participants by qualified healthcare workers. Strategies to prevent malaria infection by reducing mosquito bites were highlighted including through the use of mosquito nets. Attendees were encouraged to prevent malaria by embracing the use of treated mosquito nets and maintaining them appropriately. Participants were also advised to maintain clean environments devoid of breeding grounds for mosquitoes. Several questions on how to effectively use mosquito nets where posed by attendees and responded to by the project management team Figure 3. Page number not for citation purposes Results A total of 117 women (pregnant women and nursing mothers) were enrolled with free medical services at the three centers-Kagini (40), Gwagwa (42) and Idu-Karmo (35) Primary Health Care Centers Table 1. Health prevention talks, malaria diagnosis, provision of malaria medications and distribution of mosquito nets were ear marked for each center. Page number not for citation purposes 2 malaria using only clinical means leads to over-diagnosis [7]. In the Tanzanian study, malaria was the clinical diagnosis for 528 (60.7%), but was the actual cause of fever in only 14 (1.6%) [5]. Whereas in Namibia, only 36% of patients admitted for malaria were confirmed as malaria [6]. Beyond exposure to the adverse effects of an unwarranted antimalarial, a prospective study in Tanzania documented higher case fatality rate for slide negative, than slide positive people (p < 0.001) [8]. Reyburn and colleagues were of the view that malaria is commonly over-diagnosed in people presenting with severe febrile illness, especially in those living in areas with low to moderate transmission and in adults resulting in failure to treat alternative causes of severe infection. This failure commonly results in avoidable deaths. importance of malaria is strongly promoted; the influence of peers, conforming to perceived expectations from colleagues; pressure to conform with perceived patient preferences; and quality of diagnostic support, involving resource management, motivation and supervision [14]. Following the dictates of the guidelines-rather than "mindlines" [14] should be encouraged across all levels of care. Finally, to sustain the gains of the World Malaria Day, measures should be taken to broaden the reach to ensure sustained asses to information by the general public to prevent malaria infection in our communities, as well as parasitic diagnosis to minimize over diagnosis of malaria in infants, pregnant and nursing mothers. Authors’ contributions Obinna Ositadimma Oleribe developed the concept and reworked the initial drafts. Obison Guy Chigozie and Munaonyeso Ekweghariri developed the initial draft. Ekei Ekom, Oriaku Ofem and Chidi Igwesi led the field teams to the facilities. Grace Iyalla and Obinna Ositadimma Oleribe cleaned and analyzed the data. All authors read and approved the final manuscript. Simon David Taylor-Robinson and Obinna Ositadimma Oleribe are guarantors of the paper. The authors declare no competing interests. The authors declare no competing interests. Acknowledgments EFMC is grateful to the US Centers for Disease Control and Prevention (CDC) through the Institute for Human Virology Nigeria (IHVN) for institutional support. CFHI is grateful to National Malaria Elimination Program (NMEP) for RDTs and LTNs. SDTR is grateful to the Wellcome Trust Global Centre and to the United Kingdom National Institute for health Research Biomedical Facility at Imperial College London for funding and infrastructure support. Table and figures Table 1: Women reached, tested for malaria with RDT and given ITNs Figure 1: Map of Nigeria showing the Federal Capital Territory (FCT), Abuja where the project was implemented Figure 2: Arrival of EFMC/CFHI team at Primary Healthcare Clinic (PHC) Idu-Karmo, Abuja Nigeria Figure 3: Health talk on malaria and better health-seeking behavior, PHC Idu-Karmo Figure 4: Malaria rapid diagnostic test at PHC Idu Karimo Figure 5: Mothers displaying their ITNs at PHC Gwagwa, Abuja Table 1: Women reached, tested for malaria with RDT and given ITNs Figure 1: Map of Nigeria showing the Federal Capital Territory (FCT), Abuja where the project was implemented Figure 3: Health talk on malaria and better health-seeking behavior, PHC Idu-Karmo Figure 4: Malaria rapid diagnostic test at PHC Idu Karimo Figure 5: Mothers displaying their ITNs at PHC Gwagwa, Abuja Competing interests As clinical diagnosis has very low sensitivity and specificity due to the nonspecific nature of the symptoms and signs of malaria, this informed the development of the current malaria treatment guidelines that emphasized parasitic diagnosis rather than clinical diagnoses [9-12]. There is therefore, the need, in line with the current malaria treatment guidelines of Nigeria [9, 10], Sierra Leone [11], Liberia [12] and several other countries in malaria-endemic areas to ensure parasitic diagnosis before treatment for malaria is commenced as parasite-based diagnosis is the gold standard. Beyond diagnosis, quantification and speciation of parasites allow for improved patient care in malaria-positive patients, identification of malaria-negative patients in whom another diagnosis must be explored, avoidance of unnecessary antimalarial intake with resultant reduction in adverse effects, drug interactions and selection pressure for resistance. In addition, this will result in improved health information and proper confirmation of treatment failures. Use of RDT has its advantages which include the fact that it does not require any equipment or expertise as community health workers and field staff used it successfully, results are available within 15 to 20 minutes, can be done at the consulting room/bedside and can be used in settings where microscopy is not available. Variation in sensitivity and specificity of the various RDTs, with high levels of false negative results in those with low parasite densities and or due to prozone effect is a major drawback. Also, false positive results due to recent infection as antigen clears slowly as well as from heterophile antibodies limits the usefulness of this process. In addition, poor storage of the RDT can increase the rate of false positive or negative results as there is the need to keep the RDT cassettes/strips at low temperature. The limitations of RDT can be minimized by the use of microscopy diagnosis which is more cost effective in high transmission areas as it is cheaper, allows quantification and speciation of parasites, allows for detection of treatment failures, and allows for detection of other causes of fever and other diseases found in the area. However, microscopic diagnosis is limited by the following challenges-needs functional microscope and power source and expertise at reading stained blood smears. It is also tedious and time consuming resulting in delayed results that may also be due to high load of fever cases in the community. References The impact of the knowledge shared and rapid test carried out was evident during the field work. As severe malaria is clinically similar to other severe febrile illnesses and in endemic areas, parasitological confirmation of parasitaemia is often unavailable or unreliable, false-positive malaria microscopy is therefore very common. This most time results in wrong focus on malaria with resultant unnecessary antimalaria treatment and failure to address other life-threatening conditions [13]. Over diagnosis has been linked to the influence of initial training within a context where the 1. World Health Organization (WHO). Media Centre: Malaria Fact sheet. Updated April 2017. Accessed May 8, 2017. 1. World Health Organization (WHO). Media Centre: Malaria Fact sheet. Updated April 2017. Accessed May 8, 2017. 2. Standard Diagnostics Inc. SD BIOLINE Malaria Ag P.f: Optimal screening test for P.falciparum. Accessed May 8, 2017. Page number not for citation purposes 3. Oladosu OO, Oyibo WA. Overdiagnosis and overtreatment of malaria in children that presented with fever in Lagos, Nigeria. ISRN Infectious Diseases. 2012 Jul 19; 2013. Google Scholar 9. Federal Ministry of Health (FMOH). National Malaria Control Programme, Abuja, Nigeria . Accessed May 8, 2017 Page number not for citation purposes 4 9. Federal Ministry of Health (FMOH). National Malaria Control Programme, Abuja, Nigeria . Accessed May 8, 2017 10. FMOH. National Malaria Control Programme. Case Module 1: case management of malaria in the hospital. Trainer Manual. In press. 4. Doudou MH, Mahamadou A, Ouba I, Lazoumar R, Boubacar B, Arzika I, Maiguizo S. A refined estimate of the malaria burden in Niger. Malar J. 2012; 11: 89. PubMed \| Google Scholar 11. Government of Sierra Leone, Ministry of Health and Sanitation. National Malaria Control Policy: guide for case management of malaria. Fourth Edition. 2015. In press. 5. Crump JA, Morrissey AB, Nicholson WL, Massung RF, Stoddard RA, Galloway RL, Ooi EE, Maro VP, Saganda W, Kinabo GD, Muiruri C. Etiology of severe non-malaria febrile illness in Northern Tanzania: a prospective cohort study. PLoS Negl Trop Dis. 2013 Jul 18; 7(7): e2324. PubMed \| Google Scholar 12. Republic of Liberia. Ministry of Health and Social Welfare. National Therapeutic Guideline for Liberia and Essential Medicine Lists. 2011. Accessed May 8, 2017. 6. Van Dillen J, De Jager AJ, De Jong I, Wendte JF. Overdiagnosis of malaria in hospitalized patients in Namibia. Tropical doctor. 2007 Jul 1; 37(3): 185-6. PubMed \| Google Scholar 13. Gwer S, Newton CR, Berkley JA. Over-diagnosis and co- morbidity of severe malaria in African children: a guide for clinicians. The American journal of tropical medicine and hygiene. 2007 Dec 1; 77(6 Suppl): 6-13. PubMed \| Google Scholar 7. Njuguna J, Muita J, Mundia G. Malaria morbidity and temperature variation in a low risk Kenyan district: a case of overdiagnosis. International journal of biometeorology. 2009 May 1; 53(3): 299-304. PubMed \|Google Scholar 14. Chandler CI, Jones C, Boniface G, Juma K, Reyburn H, Whitty CJ. Guidelines and mindlines: why do clinical staff over- diagnose malaria in Tanzania: a qualitative study. Malaria journal. 2008 Apr 2; 7: 53.PubMed \| Google Scholar 14. Chandler CI, Jones C, Boniface G, Juma K, Reyburn H, Whitty CJ. Guidelines and mindlines: why do clinical staff over- diagnose malaria in Tanzania: a qualitative study. Malaria journal. Page number not for citation purposes Page number not for citation purposes 6 Page number not for citation purposes 7 9. Federal Ministry of Health (FMOH). National Malaria Control Programme, Abuja, Nigeria . Accessed May 8, 2017 2008 Apr 2; 7: 53.PubMed \| Google Scholar Table 1: Women reached, tested for malaria with RDT and given ITN Facility No of People Reached No Tested No that Received ACT No that Received ITNs Kagini 40 32 Nil 20 Idu-Karmo 35 20 8 33 Gwagwa 42 27 Nil 25 Total 117 79 8 78 Figure 1: Map of Nigeria showing the Federal Capital Territory (FCT), Abuja where the project was implemented Figure 1: Map of Nigeria showing the Federal Capital Territory (FCT), Abuja where the project was implemented Figure 2: Arrival of EFMC/CFHI team at Primary Healthcare Clinic (PHC) Idu-Karmo, Abuja Nigeria Figure 2: Arrival of EFMC/CFHI team at Primary Healthcare Clinic (PHC) Idu-Karmo, Abuja Nigeria 5 Page number not for citation purposes Figure 3: Health talk on malaria and better health-seeking behavior, PHC Idu-Karmo Figure 3: Health talk on malaria and better health-seeking behavior, PHC Idu-Karmo Figure 4: Malaria rapid diagnostic test at PHC Idu Karimo Figure 4: Malaria rapid diagnostic test at PHC Idu Karimo Page number not for citation purposes Figure 5: Mothers displaying their ITNs at PHC Gwagwa, Abuja Figure 5: Mothers displaying their ITNs at PHC Gwagwa, Abuja Page number not for citation purposes 7
https://openalex.org/W4327571614	https://link.springer.com/content/pdf/10.1007/s44229-023-00028-z.pdf	English	null	Recently Top Trending Cancers in a Tertiary Cancer Hospital in Pakistan	Dr. Sulaiman Al Habib medical journal	2,023	cc-by	5,993	Abstract Cancer is a leading cause of death, and its incidence is increasing, as reported in recent studies by GLOBOCAN. Cancer registry programs provide insights into currently trending tumors worldwide and aid in determining possible risk factors. This study was based on 7 years of cancer registry data recorded at NIMRA cancer hospital, Sindh, from 2015 to 2021. A total of 16,191 cancer patients were registered. In men, head and neck, lung, liver, colorectal and urinary tract cancers were most common. In women, breast cancer, head and neck cancer, gynecological tumors, esophageal cancer and colorectal cancer predominated. The overall data analysis indicated trending cancers in both sexes, including head and neck cancer (37.76%), breast cancer (13.83%), gynecological tumors (10.22%), esophageal cancer (5.18%), lung cancer (4.79%), colorectal cancer (4.27%), liver cancer (3.87%), lymphoma (3.16%), urinary tract cancer (3.11) and prostate cancer (1.53%). The mean age was 50.41 ± 11.78 years in men and 48.47 ± 11.88 years in women. Cancer prevalence has markedly increased worldwide, and is particularly alarming in developing countries. Various risk factors are involved in this increase, including the use of tobacco, areca nut, chewable tobacco, snuff or niswar. Current disease trends are substantially different from those in older studies at the institute. Keywords GLOBOCAN · Head and neck · Breast · Gynecological tumors · Esophagus · Lung · Colorectal · Liver · Lymphoma · Urinary tract · Prostate Abbreviations GLOBOCAN Global cancer incidence NIMRA Nuclear Institute of Medicine and Radiotherapy PAEC Pakistan Atomic Energy Commission HMIS Hospital Management and Information System HER2 Human epidermal growth factor receptor 2 HCC Hepatocellular carcinoma Recently Top Trending Cancers in a Tertiary Cancer Hospital in Pakistan F i l Ali1 S di H i 2 S jj d Ah d M 1 S d Sh hid I b l1 Received: 4 November 2022 / Accepted: 2 February 2023 / Published online: 16 March 2023 © The Author(s) 2023 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 https://doi.org/10.1007/s44229-023-00028-z RESEARCH ARTICLE * Faisal Ali faisalphy28@hotmail.com 1 Nuclear Institute of Medicine and Radiotherapy, Jamshoro, Pakistan 1 Introduction Abbreviations GLOBOCAN Global cancer incidence NIMRA Nuclear Institute of Medicine and Radiotherapy PAEC Pakistan Atomic Energy Commission HMIS Hospital Management and Information System HER2 Human epidermal growth factor receptor 2 HCC Hepatocellular carcinoma Cancer is a leading cause of mortality worldwide among people of all ages [1]. Globally, large expenditures are allo- cated to fighting against this disease. According to published research, in 2000, 6 million deaths occurred, and 22 million people were living with this deadly disease. That study pre- dicted that, on the basis of the rates at the time, 15 million new cases and approximately 10 million deaths would occur in 2020 [2]. In fact, approximately 19.3 million new cancer cases were registered, and approximately 10 million deaths were reported worldwide in 2020. According to global can- cer incidence (GLOBOCAN), the global burden of cancer is expected to be 28.4 million by 2040, a number 47% higher than that in 2020 [3]. The rapid increases in cancer incidence rates have become a serious public health issue in Pakistan and a focus of con- tinuing governmental efforts. Cancer is a multigenic and multicellular disease that can arise from all cell types [4]. In developing countries, chronic infections are responsible for causing more than 22% of cancers, and the use of betel nuts and tobacco remains the most important risk factor [5]. For future policymaking regarding cancer prevalence, 1 Nuclear Institute of Medicine and Radiotherapy, Jamshoro, Pakistan 2 Nuclear Medicine Oncology and Radiotherapy Institute, Nawabshah, Pakistan ol:.(123456789 1 3 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 43 Table 1 Registered cases from 2015 to 2021 Year Male Male (%) Female Female (%) Total 2015 1139 51 1095 49 2234 2016 1058 52 977 48 2035 2017 1119 50 1119 50 2237 2018 1204 49 1253 51 2457 2019 1215 49 1264 51 2479 2020 1159 51 1113 49 2272 2021 1214 49 1263 51 2477 Total 16,191 Fig. 2.2 Exclusion of Data Miscellaneous diseases included pathologies present in low proportions and undefined primary cases, including mela- noma, mesothelial, skin or soft tissue cases. Miscellane- ous cases were excluded from the top ten trending cancer analyses. Reported cancer cases from 2015 to 2021 are categorized according to disease in Table 2. Cases in men and women were normalized to the most prevalent disease, which was predominantly head and neck cancer, in all annual data. The normalized proportion graphs of incident disease are shown in Figs. 2, 3, 4, 5, 6, 7 and 8. Head and neck, breast and gynecological cancers had the most reported cases every year. Figure 2 shows that the top trending reg- istered cancers in 2015 were head and neck cancer, breast cancer, gynecological tumors, lung cancer, colorectal can- cer, liver cancer, urinary tract cancer, lymphoma, esophageal cancer and testicular tumors. Figure 3 shows that the top 2.1 Inclusion of Data The NIMRA cancer hospital is one of the oldest cancer hos- pital facilities built by the Pakistan Atomic Energy Com- mission (PAEC); it is located in the South Sindh province of Pakistan. Its scope includes diagnosis, nuclear medicine, and treatment of malignant cancers with chemotherapy and radiotherapy. NIMRA cancer hospital is capable of perform- ing research studies to examine current cancer trends and forecast future trends. The center is equipped with a well- established cancer registry software hospital management information system (HMIS) for data analysis. Data were registered from January 1, 2015 to December 31, 2021, and included a total of 161,191 patients with biopsies treated at NIMRA cancer hospital. Each patient was registered with an automatically generated unique ID in HMIS software. Data were categorized by year, sex and type of cancer for 7 years. The data were divided into tabulated and graphical distributions for analysis. Fig. 1 Graphical distribution of annually reported cases in both sexes The graphical distribution of the reported cancer cases by year is shown in Fig. 1. The total number of registered can- cer patients by year in Fig. 1 indicated the trends in reported cases over the past 4 years (2018–2021). The results indi- cated increased numbers of cancer patients per annum with respect to past data, except for the reported cancer cases in the year 2020 (because of the COVID-19 pandemic). 1 Introduction 1 Graphical distribution of annually reported cases in both sexes Table 1 Registered cases from 2015 to 2021 Year Male Male (%) Female Female (%) Total 2015 1139 51 1095 49 2234 2016 1058 52 977 48 2035 2017 1119 50 1119 50 2237 2018 1204 49 1253 51 2457 2019 1215 49 1264 51 2479 2020 1159 51 1113 49 2272 2021 1214 49 1263 51 2477 Total 16,191 Table 1 Registered cases from 2015 to 2021 cancer registries have been found to be a valuable source of information on associated cancer risk factors in genetic, epigenetic and epidemiological studies. Cancer demographic data captured in registries change over time, under the influ- ence of cancer screening programs; the availability of cancer treatment and diagnostic facilities; and variations in genetic, epigenetic and environmental risk factors. Epidemiological studies improve understanding of changing trends in cancer risk factors, thus aiding in interpretation and forecasting of future cancer incidence [6–9]. Cancer is a non-communi- cable deadly disease that has recently been found to be the leading cause of death [10, 11]. Asian developing countries must take immediate steps to prevent and detect cancer. Fig. 1 Graphical distribution of annually reported cases in both sexes Fig. 1 Graphical distribution of annually reported cases in both sexes 3 Results Figure 7 shows that the top trending registered cases in 2020 were head and neck cancer, breast cancer, gynecological tumors, esophageal cancer, colorectal cancer, Table 2 Normalized proportions of annually registered cases from 2015 to 2021 Site 2015 2016 2017 2018 2019 2020 2021 N Norm N Norm N Norm N Norm N Norm N Norm N Norm Bone 34 0.05 32 0.043 50 0.06 32 0.03 28 0.03 29 0.03 26 0.03 Brain 36 0.05 28 0.037 31 0.04 40 0.04 39 0.04 17 0.02 27 0.03 Breast 312 0.42 239 0.316 267 0.34 350 0.37 372 0.37 329 0.35 370 0.39 Colorectal 96 0.13 80 0.106 96 0.12 92 0.1 92 0.09 113 0.12 123 0.13 Esophagus 71 0.09 85 0.112 115 0.15 128 0.14 144 0.14 154 0.16 143 0.15 Gallbladder 17 0.02 7 0.01 19 0.02 15 0.02 13 0.01 17 0.02 15 0.02 Head and neck 748 1 756 1 779 1 947 1 1008 1 939 1 937 1 Leukemia 32 0.04 23 0.03 4 0.01 7 0.01 4 0 5 0.01 8 0.01 Liver 92 0.12 91 0.121 104 0.13 103 0.11 78 0.08 65 0.07 91 0.1 Lung 138 0.18 147 0.194 120 0.15 119 0.13 96 0.1 81 0.09 75 0.08 Pancreas 9 0.01 12 0.016 13 0.02 11 0.01 6 0.01 12 0.01 8 0.01 Prostate 27 0.04 26 0.034 38 0.05 37 0.04 50 0.05 34 0.04 36 0.04 Stomach 22 0.03 27 0.035 24 0.03 18 0.02 38 0.04 25 0.03 37 0.04 Lymphoma 77 0.1 69 0.091 78 0.1 73 0.08 78 0.08 63 0.07 73 0.08 Urinary tract 83 0.11 63 0.084 82 0.11 66 0.07 60 0.06 62 0.07 86 0.09 Testicular tumors 36 0.05 25 0.033 30 0.04 26 0.03 37 0.04 35 0.04 33 0.04 Gynecological tumors 241 0.32 202 0.267 207 0.27 248 0.26 265 0.26 225 0.24 267 0.28 Miscellaneous 163 0.22 123 0.163 180 0.23 145 0.15 71 0.07 67 0.07 122 0.13 Fig. 2 Graphical distribution of trending cancers registered in 2015 Fig. 3 Graphical distribution of trending cancers registered in 2016 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 44 trending registered cancers in 2016 were head and neck can- cer, breast cancer, gynecological tumors, lung cancer, liver cancer, esophageal cancer, colorectal cancer, lymphoma, urinary tract cancer and bone cancer. 3 Results Figure 4 shows that the top trending registered cancers in 2017 were head and neck cancer breast cancer gynecological tumors lung esophageal cancer, lung cancer, liver cancer, colorectal can- cer, lymphoma, urinary tract cancer and brain cancer. Fig- ure 6 shows that the top trending registered cases in 2019 were head and neck cancer, breast cancer, gynecological tumors, esophageal cancer, lung cancer, colorectal cancer, lymphoma liver cancer urinary tract cancer and prostate Table 2 Normalized proportions of annually registered cases from 2015 to 2021 Site 2015 2016 2017 2018 2019 2020 2021 N Norm N Norm N Norm N Norm N Norm N Norm N Norm Bone 34 0.05 32 0.043 50 0.06 32 0.03 28 0.03 29 0.03 26 0.03 Brain 36 0.05 28 0.037 31 0.04 40 0.04 39 0.04 17 0.02 27 0.03 Breast 312 0.42 239 0.316 267 0.34 350 0.37 372 0.37 329 0.35 370 0.39 Colorectal 96 0.13 80 0.106 96 0.12 92 0.1 92 0.09 113 0.12 123 0.13 Esophagus 71 0.09 85 0.112 115 0.15 128 0.14 144 0.14 154 0.16 143 0.15 Gallbladder 17 0.02 7 0.01 19 0.02 15 0.02 13 0.01 17 0.02 15 0.02 Head and neck 748 1 756 1 779 1 947 1 1008 1 939 1 937 1 Leukemia 32 0.04 23 0.03 4 0.01 7 0.01 4 0 5 0.01 8 0.01 Liver 92 0.12 91 0.121 104 0.13 103 0.11 78 0.08 65 0.07 91 0.1 Lung 138 0.18 147 0.194 120 0.15 119 0.13 96 0.1 81 0.09 75 0.08 Pancreas 9 0.01 12 0.016 13 0.02 11 0.01 6 0.01 12 0.01 8 0.01 Prostate 27 0.04 26 0.034 38 0.05 37 0.04 50 0.05 34 0.04 36 0.04 Stomach 22 0.03 27 0.035 24 0.03 18 0.02 38 0.04 25 0.03 37 0.04 Lymphoma 77 0.1 69 0.091 78 0.1 73 0.08 78 0.08 63 0.07 73 0.08 Urinary tract 83 0.11 63 0.084 82 0.11 66 0.07 60 0.06 62 0.07 86 0.09 Testicular tumors 36 0.05 25 0.033 30 0.04 26 0.03 37 0.04 35 0.04 33 0.04 Gynecological tumors 241 0.32 202 0.267 207 0.27 248 0.26 265 0.26 225 0.24 267 0.28 Miscellaneous 163 0.22 123 0.163 180 0.23 145 0.15 71 0.07 67 0.07 122 0.13 Fig. 2 Graphical distribution of trending cancers registered in 2015 Fig. 3 Results Cancer registry statistics of the reported cases for a period of 7 years (2015–2021) and the percentage incidence in both sexes are shown in Table 1. Almost on average cancer incidence reported ratio is about 50% between both sexes annually. 1 3 1 3 44 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 trending registered cancers in 2016 were head and neck can- cer, breast cancer, gynecological tumors, lung cancer, liver cancer, esophageal cancer, colorectal cancer, lymphoma, urinary tract cancer and bone cancer. Figure 4 shows that the top trending registered cancers in 2017 were head and neck cancer, breast cancer, gynecological tumors, lung cancer, esophageal cancer, liver cancer, colorectal cancer, urinary tract cancer, lymphoma and bone cancer. Figure 5 shows that the top trending registered cancers in 2018 were esophageal cancer, lung cancer, liver cancer, colorectal can- cer, lymphoma, urinary tract cancer and brain cancer. Fig- ure 6 shows that the top trending registered cases in 2019 were head and neck cancer, breast cancer, gynecological tumors, esophageal cancer, lung cancer, colorectal cancer, lymphoma, liver cancer, urinary tract cancer and prostate cancer. 3 Results 3 Graphical distribution of trending cancers registered in 2016 Table 2 Normalized proportions of annually registered cases from 2015 to 2021 Fig. 3 Graphical distribution of trending cancers registered in 2016 Fig. 2 Graphical distribution of trending cancers registered in 2015 Fig. 3 Graphical distribution of trending cancers registered in 2016 Fig. 2 Graphical distribution of trending cancers registered in 2015 esophageal cancer, lung cancer, liver cancer, colorectal can- cer, lymphoma, urinary tract cancer and brain cancer. Fig- ure 6 shows that the top trending registered cases in 2019 were head and neck cancer, breast cancer, gynecological tumors, esophageal cancer, lung cancer, colorectal cancer, lymphoma, liver cancer, urinary tract cancer and prostate cancer. Figure 7 shows that the top trending registered cases in 2020 were head and neck cancer, breast cancer, gynecological tumors, esophageal cancer, colorectal cancer, lung cancer, liver cancer, lymphoma, urinary tract cancer trending registered cancers in 2016 were head and neck can- cer, breast cancer, gynecological tumors, lung cancer, liver cancer, esophageal cancer, colorectal cancer, lymphoma, urinary tract cancer and bone cancer. Figure 4 shows that the top trending registered cancers in 2017 were head and neck cancer, breast cancer, gynecological tumors, lung cancer, esophageal cancer, liver cancer, colorectal cancer, urinary tract cancer, lymphoma and bone cancer. Figure 5 shows that the top trending registered cancers in 2018 were head and neck cancer, breast cancer, gynecological tumors, 1 3 45 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 45 Fig. 4 Graphical distribution of trending cancers registered in 2017 Fig. 5 Graphical distribution of trending cancers registered in 2018 Fig. 6 Graphical distribution of trending cancers registered in 2019 Fig. 7 Graphical distribution of trending cancers registered in 2020 Fig. 6 Graphical distribution of trending cancers registered in 2019 Fig. 6 Graphical distribution of trending cancers registered in 2019 Fig. 4 Graphical distribution of trending cancers registered in 2017 Fig. 6 Graphical distribution of trending cancers registered in 2019 Fig. 4 Graphical distribution of trending cancers registered in 2017 Fig. 7 Graphical distribution of trending cancers registered in 2020 Fig. 5 Graphical distribution of trending cancers registered in 2018 Fig. 7 Graphical distribution of trending cancers registered in 2020 Fig. 3 Results 5 Graphical distribution of trending cancers registered in 2018 232 (1.43%) bone cancer cases, 221 (1.36%) testicular tumor cases, 217 (1.34%) brain cancer cases, 191 (1.18%) stom- ach cancer cases, 106 (0.65%) gallbladder cancer cases, 85 (0.52%) leukemias, and 72 (0.44%) pancreatic cancer cases. and testicular tumors. Figure 8 shows that the top trending registered cases in 2021 were head and neck cancer, breast cancer, gynecological tumors, esophageal cancer, colorectal cancer, liver cancer, urinary tract cancer, lung cancer, lym- phoma and stomach cancer. p In men, the top trending cancers included head and neck cancer, lung cancer, liver cancer, colorectal cancer, urinary tract cancer, lymphoma, esophageal cancer, prostate cancer, testicular tumors and bone cancer, as shown in Fig. 9. Among 16,191 registered cancer cases, 8107 were in men, and 8084 were in women. Table 3 depicts the over- all distribution of 16,191 reported cases over the 7 years by sex. The aggregated cases in both sexes included 6113 (37.76%) head and neck cancer cases, 2239 (13.83%) breast cancer cases, 1654 (10.22%) gynecological tumor cases, 838 (5.18%) esophageal cancer cases, 776 (4.79%) lung cancer cases, 692 (4.27%) colorectal cancer cases, 626 (3.83%) liver cancer cases, 511 (3.16%) lymphoma cases, 503 (3.11%) uri- nary tract cancer cases, 248 (1.53%) prostate cancer cases, In women, the top trending cancers included breast can- cer, head and neck cancer, gynecological tumors, esophageal cancer, colorectal cancer, lymphoma, lung cancer, urinary tract cancer, liver cancer and bone tumors, as shown in Fig. 10. An overall analysis of the data indicated that the top trending cancers in men and women included head and neck 1 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 46 Fig. 8 Graphical distribution of trending cancers registered in 2021 available for comparison, and this was an institution-based study. Trending cancer data from our center indicated that the cancer incidence in both sexes was approximately 50%, and the predominant reported disease was head and neck cancer. Among 16,191 reported cases, the overall burden of head and neck cancers was 37.38%: 24.52% in men and 13.24% in women. The main factors contributing to these trends were the use of tobacco, areca nut, chewable tobacco, snuff, niswar, etc. [12–15]. Table 3 Classification of annual reported cases from 2015 to 2021 3 Results Areca nut is used in numerous scented forms, and betel quid (including betel leaf, areca nut, sweeteners, condiments, slaked lime, and tobacco) is used in most areas of Pakistan and is the major source of carcinogenesis for head and neck cancers [16]. Various cancers are included in the head and neck region, whereas oral cancers are the most common squamous cell carcinomas. In developing countries, compared with the Western world, squamous cell carcinomas differ in disease sites, molecular biology and risk factors. In developing countries, owing to financial limitations, poverty, illiter- acy and access to appropriate health care centers are the major barriers to diagnosis and treatment; consequently advanced stage presentation of the disease occurs in most cases [17–19]. Fig. 8 Graphical distribution of trending cancers registered in 2021 cancer, breast cancer, gynecological tumors, esophageal can- cer, lung cancer, colorectal cancer, liver cancer, lymphoma, urinary tract cancer and prostate cancer, as shown in Fig. 11. Breast cancer is diagnosed in Pakistan a decade ear- lier than in Western countries [20]. From the overall data analysis, breast cancer is the second most common cancer overall and is the first most common cancer in women in Pakistan [21]. In this study, the breast cancer incidence was 13.62% in women. Breast cancer is heterogeneous in nature, and its subtypes include luminal A, luminal B, 4 Discussion Menarche at an early age, menopause at a late age, and full-term pregnancy at a late age are associated with a modest range of risk of breast cancer development. Enhanced use of estrogen and progestogen has been linked with early menarche, late menopause, and a shortened menstrual cycle In addition the use of oral contracep Fig. 9 Top ten trending cancers in men Fig. 10 Top ten trending cancers in women Fig. 11 Top ten trending cancers in both sexes Fig. 11 Top ten trending cancers in both sexes Fig. 11 Top ten trending cancers in both sexes Fig. 9 Top ten trending cancers in men Gynecological malignancies were the third most com- mon disease in the overall reported data analysis, at 10.22%. Numerous risk factors are associated with these malignan- cies, e.g., financial instability, compromised life (living in slums, early age menarche, treatment of infertility, use of oral contraceptives, etc. Financial limitations, and a lack of education and health awareness often cause patients to visit spiritual healers rather than physicians [26]. Fig. 10 Top ten trending cancers in women Fig. 10 Top ten trending cancers in women Esophageal cancer is an aggressive lethal disease with a high mortality rate. Worldwide, approximately 90% of biopsied cancers are classified as squamous cell cancer and adenocarcinoma [27]. Epidemiology studies can help under- stand the history and risk factors, thus aiding in early diag- nosis and treatment of esophageal cancers [28]. In our study, the incidence of esophageal cancers was low, at 1.96%, in men but was 3.22% in women. The overall esophageal can- cer burden recorded in this study was 5.18%. Esophageal malignancies have alarmingly increased in women with respect to previously reported values. Esophageal cancer shows geographic variation internationally, and high and increasing rates have been observed in some areas of Asia. Environmental factors, dietary habits (nutritional factors) and the use of tobacco are the major factors associated with the development of esophageal cancer, whereas rich diets in vegetables and fresh fruits can decrease the risk [29, 30]. Fig. 10 Top ten trending cancers in women HER2 and basal-like triple-negative [22]. Reproductive factors and weight gain are associated with luminal A and luminal B [23]. Menarche at an early age, menopause at a late age, and full-term pregnancy at a late age are associated with a modest range of risk of breast cancer development. 4 Discussion The World Bank categorizes Pakistan as a low-middle- income country. Head and neck cancers are among the top ten cancers globally and are the top trending cancer reported in Pakistan. No national cancer registry data were 1 3 Table 3 Classification of annual reported cases from 2015 to 2021 Disease Male Male (%) Female Female (%) N N (%) Head and neck 3970 24.52 2143 13.24 6113 37.76 Breast 33 0.20 2206 13.62 2239 13.83 Gynecological tumors 0 0.00 1654 10.22 1654 10.22 Esophagus 317 1.96 521 3.22 838 5.18 Lung 617 3.81 159 0.98 776 4.79 Colorectal 423 2.61 269 1.66 692 4.27 Liver 499 3.09 127 0.78 626 3.87 Lymphoma 328 2.03 183 1.13 511 3.16 Urinary tract 356 2.20 147 0.91 503 3.11 Prostate 248 1.53 0 0.00 248 1.53 Bone 148 0.91 84 0.52 232 1.43 Testicular tumors 221 1.36 0 0.00 221 1.36 Brain 143 0.88 74 0.46 217 1.34 Stomach 117 0.72 74 0.46 191 1.18 Gallbladder 30 0.19 76 0.47 106 0.65 Leukemia 54 0.33 31 0.19 85 0.52 Pancreas 46 0.28 26 0.16 72 0.44 Miscellaneous 557 3.44 310 1.91 867 5.35 Total 16,191 Disease Male Male (%) Female Female (%) N N (%) Head and neck 3970 24.52 2143 13.24 6113 37.76 Breast 33 0.20 2206 13.62 2239 13.83 Gynecological tumors 0 0.00 1654 10.22 1654 10.22 Esophagus 317 1.96 521 3.22 838 5.18 Lung 617 3.81 159 0.98 776 4.79 Colorectal 423 2.61 269 1.66 692 4.27 Liver 499 3.09 127 0.78 626 3.87 Lymphoma 328 2.03 183 1.13 511 3.16 Urinary tract 356 2.20 147 0.91 503 3.11 Prostate 248 1.53 0 0.00 248 1.53 Bone 148 0.91 84 0.52 232 1.43 Testicular tumors 221 1.36 0 0.00 221 1.36 Brain 143 0.88 74 0.46 217 1.34 Stomach 117 0.72 74 0.46 191 1.18 Gallbladder 30 0.19 76 0.47 106 0.65 Leukemia 54 0.33 31 0.19 85 0.52 Pancreas 46 0.28 26 0.16 72 0.44 Miscellaneous 557 3.44 310 1.91 867 5.35 Total 16,191 Table 3 Classification of annual reported cases from 2015 to 2021 1 3 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 47 47 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42 49 HER2 and basal-like triple-negative [22]. Reproductive factors and weight gain are associated with luminal A and luminal B [23]. 5 Conclusion Liver malignancies, e.g., hepatocellular carcinoma (HCC), are the second leading cause of death globally. In Pakistan, HCC in men markedly increased in the past two decades and might become the most common malignancy in the future. The overall burden of liver malignancy was found to be 3.87%: 3.08% in men and 0.78% in women. Possible risk factors for lung malignancies are hepatitis-B and hep- atitis-C, whereas in some regions in developing countries, the disease burden due to hepatitis-B has decreased because of vaccination. A substantial number of the cases reported in tertiary care hospitals treating hepatocellular diseases are hepatitis-C positive, and hepatitis-C is a well-established risk factor for liver carcinogenesis. A lack of awareness of liver diseases among the general public has substantially increased the incidence rate in Pakistan with respect world- wide incidence. The cancer registry database and screening framework allowed us to determine possible risk factors, and support prevention and education efforts for hepatitis and HCC. Owing to the lack of availability of health education and dedicated facilities, patients with liver diseases usually present in advanced stages and have no definitive treatment options [36, 37]. Cancer is a leading cause of death globally, and its rate of incidence is increasing. Cancer trends differ between developing countries and the Western world. In this study, a substantial number of reported cases were head and neck (oral cavity) cancer, breast cancer, gynecological cancer, esophageal cancer, lung cancer, colorectal cancer and liver cancer. Various risk factors are involved in the increase in this deadly disease, e.g., use of tobacco, areca nut, chewable tobacco, snuff, niswar, etc. [12–15]. The observed disease trends have substantially changed with respect to older stud- ies from our institute [42]. Acknowledgements We thank Dr. Rameez-Ul-Islam Raja for providing valuable guidance. Author Contributions FA and SH contributed to writing the manu- script and the literature review. FA designed the study/basic concept, wrote sections of the manuscript and analyzed the data. SA and SS wrote parts of the manuscripts, proofread the manuscript and provided insights in interpretation. Funding This study did not receive any external grants from govern- ment, private or commercial sources. The overall burden of lymphomas was 3.16% in both sexes. Certain autoimmune and chronic inflammatory con- ditions, such as the immune system disorder Sjogren's syn- drome and rheumatoid arthritis, have consistently been asso- ciated with an enhanced risk of malignant lymphomas [38]. Declarations Conflict of Interest The authors declare that they have no conflicts of interest. Urinary tract infections include the urinary system, blad- der, urethra, ureters and kidneys [39]. The reported cases of urinary tract malignancies were 3.11% in both sexes: 2.20% in men and 0.91% in women. Urinary tract cancers range from small benign lesions to malignant neoplasms. Bladder malignancy is found predominantly in urinary tract cancers. Delayed diagnosis is associated with poor prognosis in the treatment of urinary tract malignancies. Early detection and follow-up pose major challenges in the management of the disease [40]. 4 Discussion Enhanced use of estrogen and progestogen has been linked with early menarche, late menopause, and a shortened menstrual cycle. In addition, the use of oral contracep- tives aggravates the risk factors of breast cancer [24]. The breast cancer prevalence is 2.5 times higher in Pakistan than in its neighboring countries. Much research remains necessary to establish cultural, socioeconomic, religious, psychosocial and psychological factors [25]. Lung cancer is a common malignant neoplasm world- wide in both sexes. The overall burden of lung cancer in this study was 4.79%: 3.81% in men and 0.98% in women. The main etiological risk factor for lung malignancies is the use of tobacco. Other factors associated with lung cancers are genetic susceptibility, inappropriate diet, air pollution and occupational radiation exposure. Lung cancer is a highly preventable malignancy and remains the most common and deadly disease globally. Comprehensive research addressing all possible risk factors in the development of lung diseases 1 3 Dr. Sulaiman Al Habib Medical Journal (2023) 5:42–49 48 must be performed to support future prevention and manage- ment efforts [31, 32]. continue to identify risk factors for prostate cancers. The known risk factors include familial and genetic factors. Understanding predictors will enable better control and prevention of the disease [41]. f In the Western world, colorectal malignancies are the fourth most common cancer. The trends in reported colorec- tal cancers in this study indicated that the overall burden of the disease was 4.27%: 2.61% in men and 1.66% in women. Possible risk factors associated with colorectal cancers include lifestyle; habits; age; chronic disease history; and mutations targeting oncogenes, genes associated with DNA repair mechanisms and tumor suppressor genes [33–35]. The top ten trending cancers accounted for 87.70% of the 16,191 total reported cases, whereas low proportion disease and miscellaneous tumors accounted for 12.30%. 5 Conclusion In regions with endemic parasites, cancer is also associated with parasitic infection. Availability of Data and Material The raw data used in this study may be requested from the authors. 1 3 References 23. Tamimi RM, et al. Traditional breast cancer risk factors in relation to molecular subtypes of breast cancer. Breast Cancer Res Treat. 2012;131(1):159–67. 1. Wu C, et al. 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W2012023351.txt	http://www.periodicos.letras.ufmg.br/index.php/caligrama/article/download/389/340	fr		Hugo lecteur de Byron: un contre-sens volontaire	Caligrama	2,004	cc-by	3,846	HUGO LECTEUR DE BYRON: UN CONTRE-SENS VOLONTAIRE' Bruno Sibona' Résumé: Il s'agit dans cet article du cornplexe systéme d'agrafes qui relie le poême de Victor Hugo 'Mazeppa', extrait de son recueil Les Orientales, au conte orienta/de Lord Byron du même nom, qui a inspiré lefeurte Hugo, et à sa traduction par Amédée Picbot, Hugo n'ayantpu lire le texte anglais. f'uiiiiserais pour ce faire une méthodologie intertextuelle et comparatiue inspirée par I'ouurage de Harold Bloom, The Anxiety of Influence et les outils tbéoriques de Gilles Deleuae et Félix Guattari tels qu'ils sont exposés dans Mille Plateaux. Ivan Stépanovitch Mazeppa (1644-1709), hetman des cosaques, héros national ukrainien du 17" síêcle, a été à plusieurs reprises une source d'ínspíratíon pour les artistes. En 1818, durant un séjour à Venise, dans un long poeme épique inspiré par un récit de Voltaire, Byron fait le récit d'un épisode de jeunesse qui aurait pu être fatal à Mazeppa. Cette aventure fut paradoxalement la cause initiale de son élévation: Mazeppa, alors page du roi de Pologne Jean Casimir V, menait à la cour une vie fort dissolue jusqu'au jour ou, surpris • Recebido para publicação em novembro de 2004 . .. Professor da City of London School, Londres. 158 CALlGRAMA, Belo Horizonte, 9: 157-169, dezembro 2004 en flagrant délit d'adultere par un noble polonais, il fut attaché nu et enduit de goudron sur un cheval sauvage et abandonné à la course furieuse de l'animal. Le cheval, né dans les déserts de l'Ukraine, y transporta Mazeppa exténué de fatigue et de faim qui fut recueilli par quelques paysans. La reconnaissance le fixa parmi ses libérateurs dont il partagea la vie inquiete et belliqueuse. Plus tard, ses qualités d'homme d'action doublées d'une intelligence subtile et d'une vaste culture le firent élire hetman, c'est-à-díre chef des cosaques d'Ukraine, três apprécié du jeune tsar Pierre le Grand. Par une tragique erreur de jugement, Mazeppa s'allia au roi de Suêde, Charles XII, dans sa guerre contre le tsar et ce retournement d'allíance consomma sa perte. Byron nous le montre en vieux général vaincu essayanr de distraire le roi épuisé en lui racontant l'histoire de sa jeunesse. Le roi s'endort, et Byron de conclure: 'What mortal his own doom may guess?' ('Que! mortel peut son destin deviner?') A la lecture du texte de Byron traduit par Amédée Pichot en 1821, Hugo s'empare du theme et compose un poeme divisé en deux parties pour ses Orientales. La premíere décrivant la course proprement dite dans toute son horreur et sa férocité fantasmatique, la deuxierne ajoutant un commentairedans leque! il compare Mazeppa au poete banni du commun des mortels pour ses excentricités, attaché sur le cheval fou de son inspiration. Mais pour finir, c'est ce voyage à travers les délires et souffrances du génie qui lui apporte la gloire: 'Sa sauvage grandeur naitra de son supplice'. Cette courte étude porte essentiellement SUl" deux aspects de la relation intertextuelle Byron-Hugo. A l'aide d'une problématique proche de celle dégagée par Harold Bloom dans son livre The Anxiety of Influence, je commencerai par examiner la citation de Byron mise en exergue par Hugo en tête de son poême et qui fonctionne comme une prétendue reconnaissance de dette SIBONA. Bruno. Hugo lecteur de Byron: un contre-sens volontaire 159 envers le poete anglais, alors que c'est avec le traducteur qu'Hugo entretient des relations intertextuelles beaucoup plus crodales. }e montreraí ensuite comment les deux volets, l'un gratesque et l'autre sublime, du texte hugolien représentent deux embranchements distincts par rapport au poême de Byron, le second volet sublime étant un commentaire métapoétique à la fois du premier volet grotesque et du texte byrannien. I Le passage d'un texte à I'autre est assuré par une erreur de rraduction, 'Away I Away I' crie le narrateur à la fin du g e chant du Mazeppa de Byron afin d'exprimer la soudaineté et la furie caractérisant le début de la course faUe. 'Away I Away " répetet-Il au début du 10 e chant, et une fois de plus trois vers plus lain. L'écho de ces mots résonne encore en deux autres accasians au début et au milieu du 11 e chant. La répétition de l'irnpératif liée à l'exclarnatíon renforce la narratíon, emportant littéralement le lecteur ou l'auditeur au loin, capturant son attention dans une dynamíque chaine de sono En répétant cinq fais le double cri, Byron utilise une technique de récitatíon qui appartient typiquement au genre épique et qui nous implique dans le rythme du galop de la chevauchée qui commence. Nous savons de sources variées que Hugo ne lisait ni ne comprenait l'anglais. Ii a lu Byron dans la traduction d'Amédée Pichot. ar, dans cette traduction, le mot 'away', qui aurait pu être traeluit simplement par 'au loin', n'apparait pas ainsi. A la fin du 11e chant de la version Pichot, au elébut du 12e et ensuite à I'imparfaít elans le 13e, nous trouvons : 'nous volons'. Une telle métaphore tout à fait absente du texte de Byron est typique du romantisme prosaíque ele Pichot qui tenel à systématiquement émousser le style acéré du poete anglais. 160 CALlGRAMA, Belo Horizonte, 9: 157-169, dezembro Hugo était tout à fait conscient de ces problêmes d'affaíblissernent du texte original et guere satisfait de .la traduction Pichot. II a certainement essayé de se référer au texte de Byron en dépit de sa propre incapacité à Iire l'anglais puisque les mots ; 'Away ! Away I' figurent en anglais comme épigraphe de son propre poeme, apres la dédicace à son ami, le peintre Louis Boulanger qui avait déjà illustré l'histoire de Mazeppa dans deux tableaux. Hugo révele ainsi la double source de son inspiration, visuelle et textuelle, mais dénie cette valeur inspirationnelle à la version française de Pichot'. Dans ses Orientales, Hugo a entrepris une nouvelle interprétation du mythe de Mazeppa plutôt qu'une translitératíon du conte de Byron qui aurait de toute façon été impossíble compte-tenu de la 'force' de Hugo telle qu'elle est définie par Harold Bloom", Le Mazeppa de Hugo allait être un contre-sens de lecture volontaíre du Mazeppa de Byron. Ceci est clairement démontré par l'appropriation (et la déformation) de la double exclamation que Hugo traduit dans son épigraphe par 'En avàht I En avant !'3. L'ídée de distance, d'espacement, a dísparuaji profit de l'idée de mouvement. Par ailleurs, au niveau rhétorique, Hugo s'approprie les contre-sens du traducteur Pichot. Dans le texte du poete anglaís; Ceci n'est guere surprenant lorsqu'on se rappelle ce qu'Hugo avaít dit au sujet de la traduction contemporaine de Shakespeare en français qu'íl préférait {le pas lire du tout de peur de Iire du Letourneur à la place. Une telle remarqlle montre bíen la méfiance d'Hugo à l'égard de toute tentative de traductíón poétique. 2 Bloom, Anxiety Influence: 'Strong poets make that history by mísreadíng another, so as to clear ímagínative spaces for themselves', p. 5, and '[oo.] really strong poets can read only themselves', p. 19. s On retrouve ici le therne hugolien de la force qui va telle que l'a Baudoín dans son analyse psychocritíque, I SI BoNA. Bruno. Hugo lecteur de Byron: un contre-sens volontaire 161 ii est certes fait plusieurs fois mention du vent produit par la vitesse de la course et au moins une fois apparaissent des ailes d'oiseaux. Ce lien est à la fois métaphorique et métonymique mais, au contraire du poêrne de Hugo dans lequel.la figure du vol est développée jusqu'à forrner une allégoríe visionnaire glabale, nulle part Byron ne dit que Mazeppa et san étalon 'volent'. Byron se montre en général parcímoníeux dans san usage de la métaphore et c'est par conséquent à partir de l'amplification - ou du contre-sens - métaphorique de Pichot (Naus volons') qu'Hugo peut se permettre de développer sa chaine textuelle. Hugo accepte I'héritage (sans toutefoís l'assumer ouvertement) et le développe par la métaphore filée du cheval ailé qui 'court' tout au long de son poêrne pour finir par aboutir au résultat poétique le plus évident : l'allégorie de Pégase. De Byron à Pichot, puis de Pichot à Hugo, naus assistons donc à un renforcement de la fonction figurative dans le texte, renforcement qui va fournir le terrain sémantique et rhétorique au commentaire métapoétique qui suit dans la seconde partie du poeme d'Hugo. II L'omissíon de Pichot et I'appropríatíon de Byron: un double déni d'ínfluence. Du point de vue de Hugo- 'poete fort' -, Byron n'étaít pas allé 'jusqu'à un certain point' ou avait 'échoué en n'allant pas assez loin', pour reprendre des expressions bloomiennes. Le Mazeppa de Hugo est né d'un possible désenchantement à la lecture du poêrne de son prédécesseur. Il s'est donc mis à écrire sa propre version dans laquelle on voit qu'íl récupere et 'reconstruit' Byron, ou plutôt l'image distordue du poete anglais, à seule fin de construire sa propre personnalité créatrice. Par rapport à son prédécesseur, Hugo agit en prédateur; la figure 162 CALlGRAMA, Belo Horizonte, 9: 157-169, dezembro 2004 de Byron et ses contes orientaux deviennent rapidement de$ éléments de son propre contexte d'auteur. Dans ce mêrne mouvement, Hugo reconnait le fait qu~ son texte, et par conséquent son inspiration, s'inscrit dans un@ chaine textuelle pré-existante. Mais dans ce processus utt maillon a disparu : Hugo a soigneusement évité toute référence explicite à la traduction de Pichot. Quelle que soit la qualit€ littéraire ( ou plutôt son manque de qualité si l'on en croit à la fois Byron et Hugo) de ce chainon manquant, la signification dê son omission doit être pleinement appréciée. Une re-lecture de la traduction Pichot nous prouve qu'effectivement Hugo enitt retiré une majeure partie de son inspiration, notarnment POUl." sa premiere partie, dans laquelle il s'agit souvent d'une vérítablê ré-écriture versifiée. En réalité, l'interprétation hugolienne du texte de Píchot fonctionne non seulement par amplification hyperbolique et accroissement de la figuralité, mais aussi, dans une certaine mesure, par déplacements et condensations comme dans le travaíl du rêve décrit par Preud", II est donc possible de considérêr -, Sigmund Freud, chap.VI 'The Dream-work', in The Interpretation of Dreams, trans, by James Strachley (London: Penguin Books, 1976): 'Each elements ofthe dream's content turns out to have been "overdetermíned" [...]. Not only are the elements of a drearn determined by the dream-thoughts many times over, but the individual dream-thoughts are represented in the dream by several elernents [...]. The elements of the drearn are constructed out of the whole mass of dreamthoughts and each one of these elements is shown to have been determined many times over in relatíon to the dream-thoughts', pp. 388-389. Also, 'It thus seems plausíble to suppose that in the dream-work a psychical force is operating which on the one hand stríps the elements which have high psychical value of their intensity, and on the other hand, by means of overdetermination, creates from elements of low psychical value new values, which afterwards find theír way ínto the drearn-content. If that is so, a transference and displacement ofpsycbical intensities occurs in the process of drearn-formatíon, and it is a result of these 51 BONA, Bruno. Hugo lecteur de Byron: un contre-sens volontaire 163 cette activité et ces techniques d'appropriation, de transformations, de condensations comme três proches de ce que Bloom appelle clinamen et tesserai, Esteve nous montre comment Hugo a utilisé le texte de Pichot: Tout cela Victor Hugo l'a pris à son modele, mais il le resserreà sa guise, le réfracte à travers son imagination, l'accommode à son dessein. L'interprétation symbolique lui appartient en propre. Elle a surgi à ses yeux sous la forme antithétique, entre les lignes de la traduction Pichot [... ]. II a ajouté au texte du poete anglais dans le premier cas [Mazeppa] une idée [... ]. En imitant, il invente": (c'est moi qui souligne) On voit dans cette citation une certaine confusion entre le texte original de Byron et la traduction de Pichot. La distinction that the difference between the text of the drearn-content and that of the dreamthoughts comes about [...[, The consequence of the displacement is that the drearncontent no longer resembIes the core of the dream-thoughts and that the dream gives no more than a dístortíon of the dream-wísh which exists in the unconscious', p. 417. 5 '1. Clinamen, which is poetic misreading ar mlsprísíon proper [...]. A poet swerves away from his precursor, by reading his precursor's poem as to execute a cIinamen in a reIation to it. This appears as a corrective movement in his own poem, which implies that the precursor poem went accurately up to a certain point, but then should have swerved, precisely in the direction that the new poem moves. 2. Tessera, which is completion and antithesís; I take the word not from mosaíc-rnakíng, where it is stilI used, but from the ancient mystery cuIts, where it meant a token of recognition [...]. A poet antitheticaIIy "completes" his precursor, by so reading the parent-poern as to retain its terms but to mean them in another sense, as though the precursor hacl failed to go far enough', Bloom, Anxiety Influence, p. 14 , Esteve, Byron, p. 304. 164 CALlGRAMA, Belo Horizonte, 9: 157-169, dezembro 2004 n'est pas c1airement établie entre ce qui revient à l'un et à l'autre dans l'influence exercée sur Hugo. Notre tâche est maintenant d'arriver à lire entre ces lignes, ou, plus précisément, entre celles du texte original anglais, celles de sa traduction en français et celles de la ou des versions de Hugo. Esteve a remarqué que Hugo a pris modele sur le texte de Pichot mais qu'ille 'resserre à sa guise, le réfracte à travers san imagination, l'accommode à son dessein'. Ce 'resserrement' et cette 'réfraction' sont similaires aux mécanismes de condensation et de déplacement actifs dans le travail du rêve, mais cette fois bien súr au niveau intertextueF. Le texte du prédécesseur est ainsi analogue au stock d'images et de symboles inconscients qui nourrissent les rêves du sujet", L' 'accommodement' peut être observé dans les nombreux développements hyperboliques qu'opêre Hugo sur certains motifs particuliers. III Le 'dépliement' des couches textuelles. ]'aimerais maintenant approfondir l'analyse du systême d'accroches qui relie les textes les uns aux autres. Lorsque naus commençons à lire le poême d'Hugo, naus sommes immédiatement frappés par le premier mot du texte Iuí-même, l'anacoluthe 'Ainsi'. Ce mot fonctionne comme une marque de reconnaissance d'un texte précédent (ou également d'un tableau, ne l'oublions pas). Cet adverbe représente pour le lecteur le Son inspiration plus personnelle interviendrait ainsi en partie comme un prisme distordant, le type de phénornêne qu'à la suite du critique Jean-Pierre Richard j'aimerais appeler 'diffraction' ou 'réverbération'. a Ce parallélisme me parait par ailleurs légitimé par la comparaison extensive entre les processus elu rêve et ceux ele la création poétique qu'a établi Hugo luimême et qu'il a décrit de mantere plus approfonelie elans son texte 7 Promontorium somnii. SIBONA, Bruno. Hugo leeteur de Byron: un contre-sens volontsire 165 bord mêrne de la tessera tandis qu'en mêrne temps ii autorise Hugo à libérer san prapre texte des événements qui précédent le début de la folle chevauchée dans le conte de Byron. Cet 'Ainsi' naus place donc immédiatement et de plain-pied dans le véritable Iieu symbolique ou tout se joue. En corollaíre, un blanc est laissé à rernplir au lecteur qui doit Iíre la version Byron/Pichot s'il veut comprendre comment Mazeppa en est arrivé là. Bien SUl', la vaste rnajorité des lecteurs français contemporains connaissaient la traduction de Byron par Pichot (le plus grand succês d'édition de la premiere moitié du 19 c síecle), ou étaient familiers de l'histoire grâce aux nombreuses représentations píctúrales et au spectacle du cirque équestre Franconi. De maníere díscrête mais efficace, ce vide référentie1 fonctionne donc comme une autre marque de reconnaissance, de la part de Hugo, de son inscription dans une chaine textuelle qui lui pré-existe. Le poeme de Hugo est composé de deux parties. La premiere, la plus longue, formée de 17 straphes, suit assez fídelernent la narration de la chevauchée frénétique qui cornmence au chant 9 chez Byron et au chant 11 chez Pichar'. Seules les deux derniêres strophes de la prerníere partie de Hugo font écho au commentaire byronien SUl' la difficulté de déchiffrer la destinée humaine. La seconde partie du poêrne, avec seulement six strophes, propose un cornmentaíre métapoétique de la prerniêre partie. De maniere tout à fait frappante, cette seconde partie commence également avec l'adverbe 'Ainsi' qui fonctionne maintenant cornrne référence anaphorique à la premíere partie. Tout à fait intentionnellement, les trais premieres lignes de la seconde partie se présentent donc comme des anamorphoses syntactiques et sémantiques des trais premieres lignes de la prerniere : s Effectívement, Pichot n'a pas respecté le découpage en chants de Byron. 166 CALlGRAMA, Belo Horizonte, 9: 157-169, dezembro 1c Aínsí, quand Mazeppa, qui rugit et qui pleure, Avu ses bras, ses pieds, ses flancs qu'un sabre effleure, Tous ses membres líés [... ] 2' Ainsi lorsqu'un mortel, sur qui son dieu s'étale S'est vu lié vivant sur ta croupe fatale, Génie ardent coursier [... ] Le contenu métapoétique est induit par un seul mot, 'génie', dont on connait l'importance pour Hugo et qui représente le véritable (et double) clinamen entre la premiêre partie narrative et son commentaire, mais aussi entre l'interprétation explicite de Byron sur l'histoire de Mazeppa et la lecture personnelle qu'en fait Hugo. Par conséquent, ce n'est plus trois versions de l'histoire de Mazeppa que nous devons comparer et mettre en perspective, mais bien quatre ! En effet, les deux parties du poerne de Hugo fonctionnent sur deux modes d'interprétations différents du mythe textuel tel qu'il avait été transmis par Byron: d'abord une mise en scêne fictionnelle, Pl1is un commentai.re poétíco-díscursif Ces deux versions hugolíennes doivent être examinées séparément. Dans sa premiere partie grotesque, Hugo accorde autant d'importance à certains motifs présents dans la version Pichot, comme par exemple le passage initial avec les bourreaux qui correspond également au tableau de Boulanger appartenant à Hugo à l'époque. D'autres motifs sont mentionnés mais voient leur importance réduite, tella traversée de la rivíere, ou déplacés, tel le passage relatif aux chevaux sauvages. Cependant.xle déplacement effectué par Hugo sur certe scene-cí, et le faít que ces chevaux maintenant suivent Mazeppa et son étalon au lieu d'assister à la mort de l'étalon, opere une condensation telle que nous l'avons formulée auparavant. Le rythme hypnotíque SIBONA. Bruno. Hugo lecteur de Byron: un contre-sens volontaire 167 du galop est aussi maintenu grâce à de nombreuses répétitions. La vision cosmique du ciel, du soleil globulaire, de la succession des nuits et des jours est soigneusement respectée. Enfin deux aspects importants sont considérablement développés dans de nouveaux groupements d'images : Le premier est le double motif du liquide et du feu, déjà présent dans la version Byron-Pichot, mais fortement accentué chez Hugo, peut-être parce que ce motif revêt de puissantes connotations sexuelles, aux résonnances sado-rnasochístes. Le deuxieme, sans doute le plus important, est le theme du vol dérivé du 'Nous volons' de Pichot, alors qu'il était à peu pres absent dans l'original de Byron. Hugo l'introduit à deux reprises dans sa premíere partie, avant d'en faire une exploitation systématique dans sa seconde. II y devient d'ailleurs le thême dominant, couplé avec celui de la visiono lei, nous voyons que Hugo a formé un theme supplérnentaíre à partir de deux éléments différents de la version Byron-Píchot qu'il a pu fondre grâce à son extraordinaire alchimie verbale. II a lié ce motif de la vision, crucial pour la compréhension de sa propre poétique, au thêrne de la course à partir de la séquence verbale 'Ils vont', 'IIs volent'. II opere ainsi une sorte de glissando phonique et sémantique tout au long de son poeme. Dês le début de la premíere partie, Hugo place Mazeppa dans la position du témoin de son propre tourment à partir du sens de la vue : 'Mazeppa qui rugít et qui pleur/a vu [... ]'. La vue est ainsi le sens premier à l'aide duquel le héros reste en contact avec la réalité. lmmédiatement aprês l'essor initial de la course, exprimé par un fulgurant cri métonymique fusionnant tous les acteurs ensemble, les bourreaux, le cheval et Mazeppa luímême, 'Un cri part', Hugo introduit le thêrne du vol ; 'volent avec les vents'. Si le rythme de la course est donné par la répétition au début des strophes 4 et 5 de l'expression reprise 168 CALlGRAMA, Belo Horizonte, 9:157·i69, dezembro 2004 de Pichot 'l1s vont', au début de la strophe 7, la formule 'il voit' lui faít écho, et c'est comme si l'homophonie 'vu', 'volent', 'vont', 'voit' exemplifiait l'équation de la vision au vol, du 'voír' au 'voler' à partir de l'action catalytique du 'va', de l'aller'". La configuration voir-aller-voler s'établit donc comme le lien thématique majeur entre les deux parties du poerne de Hugo, et nous pouvons maintenant mieux comprendre comment la deuxíême partie sublime peut fonctionner en tant que diffraction à la foís de la premíere partie et de la version Byron-Pichot. La diffraction de signífícatíon entre les deux parties est considérable. Cependant, la seconde partie fonctionne comme une image-miroir de la premiere. En tant que telle, elle en renforce la dimension symbolique. L'hístoíre initiale ne produirait aucune signification métapoétique sans sa contre-partie spéculaire et demeurerait purement imaginaire, une allégorie sans aucune def pour I'ínterpréter, Grâce à cette structure binaire inscrite dans le tissu textuel de son poeme, Hugoajoute une nouvelle perspective à l'histoire telle qu'elle est narrée par Byron, développant et perpétuant ainsi le mythe littéraire de Mazeppa. Resumo: Este trabalho trata do complexo sistema de relações entre o poema de Victor Hugo "Mazeppa", extraído da coletânea Les Orientales, ao conto oriental de Lord Byron de mesmo nome, o qual inspirou o jovem Hugo, e a sua tradução por Amédée Picbot, uma vez que Hugo não pôde ter lido o texto em inglês. Será utilizada a metodologia intertextual e comparativa inspirada pela obra de Harold Bloom, The Anxiety of Influence, bem como os conceitos teôricos de Gilles Deleuze e Félix Guattari expostos em Mille Plateaux lO Et 1'00 peut bien entendu penser icí à la 'force qui va' de Charles Baudoin, 51BONA. Bruno. Hugo lecteur de Byron: un contre-sens volontaire 169 Referências bibliográficas BAUDOIN, Charles, Psycbanalyse de Victor Hugo. Paris: Armand Colin, 1972. BLOOM, Harold, The Anxiety of Influence, a Theory of Poetry. Oxford: Oxford University Press, 1973. BYRON, George G., Lord, Oeuures completes, traduites de l'anglais par MM. A.[médée] P.[ichot] et E.[usebe] D.[e] S.[alle], troisieme édition, 15 vols. Paris: Ladvocat, 1821. BYRON, George G., Lord, Works, ed. by j'erome ]. McGann. Oxford: Oxford University Press, 1986. ESTEVE, Edmond, Byron et le romaruisme français. Paris: Boivin, 1907. FREUD, Sigmund, The Interpretation of Dreams, in Complete Works, trans. by James Strachey, The Standard Edition, 24 vols.London: The Hogarth Press, 1955, IV FREUD, Sigmund, The Interpretation ofDreams, transl. by James Strachey, 15 vols. London: Penguin Books(fhe Pelican Freud Library, 1976, IV HUGO, Victor, Oeuures completes/ Critique, ed. par Jean-Pierre Reynaud. Paris: Laffont/Bouquins, 1985. HUGO, Victor, Oeuures poétiques I & II, ed. par Pierre Albouy. Paris: Gallimard/Pléíadc, 1967. HUGO, Victor, Les Orientales, ed. par Pierre Albouy. Paris: Gallimard/NRF, 1964. RICHARD, Jean-Pierre, Poésie et profondeur, Paris: Seuil, 1955. VOLTAIRE, Histoire de Charles XII, roi de Suêde, in Oeuures bistoriques, ed. par René Pomeau. Paris: Gallimard/Pléíade, 1957.
https://openalex.org/W4298120165	https://ijcsm.springeropen.com/counter/pdf/10.1186/s40069-022-00554-4	English	null	Modified Artificial Neural Networks and Support Vector Regression to Predict Lateral Pressure Exerted by Fresh Concrete on Formwork	International journal of concrete structures and materials	2,022	cc-by	11,642	© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Abstract In this study, a modified Artificial Neural Network (ANN) and Support Vector Regression (SVR) with three different opti- mization algorithms (Genetic, Salp Swarm and Grasshopper) were used to establish an accurate and easy-to-use mod- ule to predict the lateral pressure exerted by fresh concrete on formwork based on three main inputs, namely mix proportions (cement content, w/c, coarse aggregates, fine aggregates and admixture agent), casting rate, and height of specimens. The data have been obtained from 30 previously piloted experimental studies (resulted 113 samples). Achieved results for the model including all the input data provide the most excellent prediction of the exerted lateral pressure. Additionally, having different magnitudes of powder volume, aggregate volume and fluid content in the mix exposes different rising and descending in the lateral pressure outcomes. The results indicate that each model has its own advantages and disadvantages; however, the root mean square error values of the SVR models are lower than that of the ANN model. Additionally, the proposed models have been validated and all of them can accurately predict the lateral pressure of fresh concrete on the panel of the formwork. Keywords: ANN, SVR, machine learning, lateral pressure, mix proportion, concrete, formwork labour work in the field. Furthermore, SCC increases the potential of construction productivity, reduces the total cost of the structure, provides a sustainable environment, and improves the quality of the structures (Almeida Filho et al., 2010). In general, there are many studies on the per- formance of SCC, such as mechanical strength (Roussel & Cussigh, 2008), durability (Tabatabaeian et al., 2017) as well as cost and life cycle assessment (Margallo et al., 2015). Development of SCC was undertaken by authors Okamura (1997) and Okamura and Ozawa (1996) in Japan. Despite having abundant research on the benefits of SCC, related to its performance and flow of SCC since its first developments in the late 1980s in Japan, there are many limitations and a lack of results regarding the expected lateral pressure exertion on the different types of formwork (Shakor & Gowripalan, 2020). Modified Artificial Neural Networks and Support Vector Regression to Predict Lateral Pressure Exerted by Fresh Concrete on Formwork Amirreza Kandiri1, Pshtiwan Shakor2,3,4* , Rawaz Kurda5,6,7 and Ahmed Farouk Deifalla8 International Journal of Concrete Structures and Materials International Journal of Concrete Structures and Materials Kandiri et al. Int J Concr Struct Mater (2022) 16:64 https://doi.org/10.1186/s40069-022-00554-4 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 https://doi.org/10.1186/s40069-022-00554-4 1 Introduction Generally, conventional concrete has an exerted pres- sure on formwork at an early stage of casting. Specifically, self-compacting concrete (SCC) type is a novel type of concrete that has the ability to flow quickly without any effort. This type of concrete has high exerted lateral pres- sure on the formwork because of its high flowability. It can fill the formwork and compact under its own weight which eliminates the shaking and vibration process through vibrator. This also reduces noise pollution and Journal information: ISSN 1976-0485 / eISSN 2234-1315 *Correspondence: pshtiwann@yahoo.com; pshtiwan.shakor@iconsmat.com. au 2 Technical College of Engineering, Sulaimani Polytechnic University, Sulaymaniyah 46001, Iraq Full list of author information is available at the end of the article 2 Technical College of Engineering, Sulaimani Polytechnic University, Sulaymaniyah 46001, Iraq Full list of author information is available at the end of the article 2 Research Significance SCC has many advantages in terms of fresh state, but it may generate high lateral pressure on the formwork that has not been studied in detail. Thus, this research aims to provide an approach to estimate the maximum lateral pressure of concrete (conventional and self-compacting concrete). Guessing the behaviour of Bingham fluid (e.g. concrete) would be interesting which is part of input and output data to produce the right models and clos- est approach to the resulted outcome. Concrete at the hardened stage might be easier to predict considering the complexities of the mixed components of concrete at the early ages. Predicting the lateral pressure of fresh con- crete is highly important and considered a great outcome to know its lateral pressure before pouring. On the other hand, there are not enough studies on predicting the lat- eral pressure of concrete on the formwork and there are no investigations particularly on the machine learning modelling to predict the lateral pressure of fresh concrete on the formwork. The predicting models could be pre- cise for engineering application predictions and it could eliminate the need for extensive further laboratory in situ testing (Vickers, 2017), and it will be advantageous for the design of the formwork. Machine learning techniques such as Artificial Neural Network (ANN) and Support Vector Regression (SVR) have been used to predict concrete properties in the past two decades (Ahmadi et al., 2020; Alam & Al Riy- ami, 2018; Farooq et al., 2020; Kandiri & Fotouhi, 2021; Kandiri et al., 2020; Lizarazo-Marriaga et al., 2020; Mohammed et al., 2021; Ramezani et al., 2020; Velay- Lizancos et al., 2018; Vickers, 2017; Yu et al., 2020). For instance, Golafshani et al. (2020) modified ANN with a multi-objective grey wolf optimizer and used that to forecast the compressive strength of high-performance and normal concrete (Golafshani et al., 2020). The study of Kandiri et al. (2021) used three different optimiza- tion algorithms to modify ANN and used those models to predict the compressive strength of recycled aggregate concrete (Kandiri et al., 2021). In another study, Jahangir and Eidgahee (2021) used a hybrid artificial bee colony algorithm-ANN to evaluate the bond strength of fibre- reinforced polymers-concrete. In addition, Ahmed et al. predicted 7 and 28 days of flexural strength of normal concrete using SVR (Jahangir & Eidgahee, 2021). Page 2 of 22 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 SCC is highly subject to changes in material behav- iour and characterization (Kurda et al., 2022); there- fore, it is necessary to have a suitable quality control. One way to overcome this is by controlling the fresh properties of concrete that directly affect its lateral pressure on the formwork. To ensure adequate fresh properties, it is necessary to have a suitable SCC mix. These are also substantial investigations of lateral pres- sure properties in the fresh SCC, hardened properties, and durability (Brito & Kurda, 2021; Koehler, 2007). Since a number of parameters affect the performance of concrete and its lateral pressure on the formwork (Gowripalan et al., 2021), it is necessary to use machine learning on the SCC and conventional concrete as well (Shakor & Pimplikar, 2011). Having limited data on the SCC lateral pressure causes difficulty in simu- lating and predicting lateral pressure on formwork by machine learning; therefore, studies on lateral pressure of conventional concrete have been included in the data collection.i study, the Genetic Algorithm (GA), Salp Swarm Algo- rithm (SSA), and Grasshopper Optimization Algorithm (GOA) are used to develop a systematic approach to achieve the optimum structures and increase the accu- racy of the models as much as possible. 3 Methodologyl As shown in the flowchart (Fig. 1), this study was made to estimate the lateral presser exerted by fresh concrete on the formwork, namely by using modified ANN and SVR. For that purpose, data from 30 previous studies (113 experimental data) were collected (Appendix 1). As input data, (i) mix composition such as cement, coarse aggre- gates, fine aggregates, water and admixture contents was considered as well as (ii) dimensions of casted specimen and (iii) casting rate of concrete (Sect. 3.1). Nevertheless, the performance of SVR and ANN sig- nificantly depends on the selection of training set data and features. These data are not only used for SCC, but also applicable for three-dimensional printed concrete as shown in the study (Izadgoshasb et al., 2021). The num- ber of hidden layers and their nodes for ANN, and the values of the penalty coefficient, error limit, and the slack variable for SVR determine the models’ performance. However, the challenge is in the fact that it is needed to determine these parameters in the first step, and they are not changeable during the process. Therefore, in this Two most accurate algorithms were considered to establish the required model based on the collected data, namely SVM (Sect. 3.2) and ANN (Sect. 3.3). However, accurate mathematical methods cannot solve optimi- zation problems. Hence, to find the optimum answer possible at a convenient time, heuristic and metaheuris- tic algorithms are used. For that purpose, GA, SSA and GOA are used (Sect. 3.4) in order to modify the SVM and ANN models. The modification process of the two algo- rithms are shown in Sect. 3.5. Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 3 of 22 ig. 1 The flowchart diagram process followed in this study. Fig. 1 The flowchart diagram process followed in this study. Page 4 of 22 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 3.1.2 Fresh Properties of Concrete Concrete at early ages, called fresh-state concrete, behaves as a Bingham fluid. This fluid is quite different from water which is called Newtonian fluid. Bingham fluid is commenced with a yield stress point (YP) which is basically the starting point of the concrete rheology. Concrete materials behave as a thixotropic material with a dilatant property (shear thickening) (Feys et al., 2009). Fig. 4 shows the comparison between Bingham fluid and Newtonian fluid. These factors assist in evaluating and predicting the lat- eral pressure of the fresh concrete on the formwork using each model as statistical evaluation parameters. These would be also beneficial for the construction industry to identify and predict the value of the pressure before the casting process. Table 3 (Appendix 1) represents the results of earlier studies on exerted lateral pressure of concrete, mix design, casting rate, slump and height of casting. l Shear thickening materials are such materials that the viscosity increases with increasing of the shear rate. Generally, it has a resistance to the applied rate when it applies. However, these could be beneficial for concrete to reduce its lateral pressure at a later stage. Based on the available data in Table 3 (Appendix 1), the data could be entered and applied into algorithm model- ling for machine learning. The selected algorithms have been chosen and attempted for the process and the most suited are opted for the application. Regarding the formwork and instalment of the form- work panel, on the construction site; it is required to provide a high amount of bracing to prevent any collapse due to huge pressure on the lateral side of the formwork. Regarding the formwork and instalment of the form- work panel, on the construction site; it is required to provide a high amount of bracing to prevent any collapse due to huge pressure on the lateral side of the formwork. However, this extreme pressure on the formwork will increase when the height of the casting structural mem- ber increases due to the gravitational unit and height rate. Fig. 5 displays certain details on the normal form- work that should be considered during casting and brac- ing it properly before casting. 3.1 Materials and Data Collectionh mixes made with w/c of 0.3–0.9. The most common mix design ratio also for concrete proportion is the 1:2:4 mix ratio which represents the cement ratio 1 to fine aggre- gate ratio 2 and the coarse aggregate 4. However, other mix designs could be observed in the matrix of concrete mixes, such as 1:1.25:2.25, and 1:2.25:3.75. The experimental dataset consists of 113 data extracted from 30 previous studies, which is tested to provide mod- els from both types of concrete. This study counted dif- ferent factors in its approaches, for example, the height effect of the casted specimens, the casting rate (place- ment rate) of casting the concrete, the constant gravita- tional unit, the ratio of water to cement, the quantity of cement, coarse aggregate, fine aggregate, and admixtures. Fig. 2 illustrates histograms of the inputs and the output of the models. Moreover, Table 1 represents the statisti- cal parameters of the inputs and the output.h P = ρ×g×H, 3.1.1 Mix Composition of Concrete Concrete mix design uses various methods to mix and various systems to cure them. Most of the concrete mixes are considered to be evaluated through visual inspection (Day, 2006). According to the earlier studies by Alyamac and Ince (2009), the concrete mix design could be com- bined their expression of fresh and hardened into one graph in terms of compressive strength. These could be presented in a nomogram diagram; see Fig. 3. Sometimes the formwork and handling procedure would be so costly as to exceed the total cost of concrete implementation by 40% (Haron et al., 2005; Lloret et al., 2015; Shakor & Gowripalan, 2020). Therefore, consider- ing to have a better implementation of casting concrete and to not lose the formwork and the proper amount of concrete, it is better to understand and study more on exerted lateral pressure modelling to mitigate the risk of losing the cost of implementation and prevent the risk of injury at the field. However, as Fig. 3 shows that the w/c ratio directly affects the compressive strength, with increasing w/c ratio is decreasing the compressive strength. On the other hand, the increase in the w/c ratio increased the workability and lateral pressure on the formwork as a result. Therefore, it should be considered that the lateral pressure highly depends on the percentage of water con- tent in the mix design. i However, according to the theoretical calculations, the lateral pressure of concrete on formwork depended on height which is related to the rate of the gravita- tional unit. Moreover, the density of materials relatively depends on the mass of the materials. Therefore, accord- ing to the law of hydrostatic pressure, the maximum pres- sure could be expressed as below Eq. (1) (Merriam, 1992): In the collection of data shown in Appendix 1, the w/c ratio is between 0.3 to 0.9. It is quite obvious that the maximum water content in the mix design of concrete is usually 0.6–0.7 of cement ratio. However, sometimes the w/c ratio may be changed in the construction site in order to achieve a certain workability. This kind of changes must not be allowed. In other words, the out- put of this study can be recommended only for concrete P = ρ×g×H, (1) Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 5 of 22 Fig. 2 Histograms of the input and the output variables. Fig. 3.1.1 Mix Composition of Concrete 2 Histograms of the input and the output variables. Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 6 of 22 Table 1 Descriptive statistics of the input and output variables. Cement (kg/m3) Water (W/C) Coarse aggregates Fine aggregates Admixture (ml/100 kg) Total height (m) Casting rate (m/h) Maximum pressure (kPa) Maximum 500 1 2400 1200 10 17 71 186 Minimum 182 0 305 379 0 0 0 2 Mean 388 1 1008 799 1 3 7 48 Standard deviation 82 0 459 182 2 3 15 37 Kurtosis 0 3 4 0 7 7 14 3 Skewness 0 2 2 0 3 2 4 2 Fig. 3 Mix design nomogram of water-to-cement ratio in relation to compressive strength as design criterion (Alyamaç & Ince, 2009; Monteiro et al., 1993). Table 1 Descriptive statistics of the input and output variables. Cement (kg/m3) Water (W/C) Coarse aggregates Fine aggregates Admixture (ml/100 kg) Total height (m) Casting rate (m/h) Maximum pressure (kPa) Maximum 500 1 2400 1200 10 17 71 186 Minimum 182 0 305 379 0 0 0 2 Mean 388 1 1008 799 1 3 7 48 Standard deviation 82 0 459 182 2 3 15 37 Kurtosis 0 3 4 0 7 7 14 3 Skewness 0 2 2 0 3 2 4 2 Table 1 Descriptive statistics of the input and output variables. Fig. 3 Mix design nomogram of water-to-cement ratio in relation to compressive strength as design criterion (Alyamaç & Ince, 2009; Monteiro et al., 1993). Fig. 3 Mix design nomogram of water-to-cement ratio in relation to compressive strength as design criterion (Alyamaç & Ince, 2009; Monteiro et al., 1993). Fig. 4 Bingham fluid behaviour compared with Newtonian fluid. where P is pressure, ρ is density, g is gravity and H is the height of the concrete. According to Puente et al. (2010), the lateral pressure of fresh concrete on the formwork is theoretically expressed as (Eq. 2): (2) P = c × γ × H, (2) where P is lateral pressure, λc is the relationship between horizontal and vertical pressure, γ is the concrete weight and H is the height of the concrete. However, Rodin (1952) explained that the maximum pressure Pmax could be found when the concrete mix is 1:2:4 with a slump of 150 mm and temperature is about 21 °C and density is assumed to be about 2400 kg/m3 (Eqs. 3.1.1 Mix Composition of Concrete , n yi − b + W TXi ≤ε + δ− i , i = 1, 2, . . . , n b + W TXi −yi ≤ε + δ+ i , i = 1, 2, . . . , n 3.1.1 Mix Composition of Concrete 3, 4): (3) Hm = 1.63 × R0.5, Hm = 1.63 × R0.5, (3) Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 7 of 22 Fig. 5 Schematic illustration of the erecting formwork. Fig. 5 Schematic illustration of the erecting formwork. into a higher-dimensional feature space with linearly sep- arable output data: (4) Pmax = 23.4 × Hm, Pmax = 23.4 × Hm, (4) where Hm is the height which the maximum lateral pres- sure occurred which is considered to be (m), Pmax is the maximum lateral pressure of the concrete on the form- work in (kPa) and R is the casting rate of the concrete (m/h). These dataset and earlier information on the con- crete pressure and different mix designs would be benefi- cial to create a model to predict the lateral pressure of the concrete on the formwork and find the maximum pres- sure on the formwork panel.h (5) Y = W TX + b, Y = W TX + b, (5) where W is a multi-dimensional vector determining the orientation of the hyperplane, and b is the bias term. Support vector regression (SVR) is a machine learn- ing method, which was proposed in 1992 (Boser et al., 1992). It has been used to solve non-linear classifica- tion, regression, and prediction problems recently (Ahmad et al., 2020; Suykens & Vandewalle, 1999; Vap- nik, 2013). The following equation represents the SVR’s objective function (Eq. 6): Therefore, usually expect the lateral pressure of con- crete should be one of the following expectation for the concrete. This is based on the materials mix, density, temperature and slump. Fig. 6 displays all types of lat- eral pressure of concrete on formwork. However, Rodin (1952) proposed that the formwork should be designed for full hydrostatic pressure considering the density of concrete. (6) minw,b 1 2w2 + C n i=1 δ+ i + δ− i , (6) s.t. b + W TXi −yi ≤ε + δ+ i , i = 1, 2, . . . , n yi − b + W TXi ≤ε + δ− i , i = 1, 2, . . . , n δ+ i ≥0, δ− i ≥0, i = 1, 2, . . . , n b + W TXi −yi ≤ε + δ+ i , i = 1, 2, . . . 3.2 Support Vector Regression Support vector machine (SVM) is an AI-based method that is used for classification and regression analysis using hyperplane classifiers. The best hyperplane can minimize the risk of classification by maximizing the distance between the two classes, where the support vec- tors lie (Sun et al., 2019). A linear regression hyperplane equation is defined as Eq. (5) by mapping the input data yi − b + W TXi ≤ε + δ− i , i = 1, 2, . . . , n δ+ i ≥0, δ− i ≥0, i = 1, 2, . . . , n Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 8 of 22 Fig. 6 Lateral pressure exerted on formwork at different points (Puente et al., 2010). This method is developed for linear classification; therefore, to solve non-linear classification kernel func- tions are defined (Brereton & Lloyd, 2010). Any func- tion which is symmetric, positive and semi-definite (Mercer’s condition) qualifies to be a kernel function (Pan et al., 2009). However, the most used one is the Gaussian radial basis function (RBF). The description of the function is defined in Eq. (9) (Smola & Schölkopf, 2004; Vapnik et al., 1997):. δ+ i δ− i = 0, where C is the penalty coefficient, ε is the error limit, δi is the slack variable above the sample observation point and δ ′ i is the slack variable under the sample observation point. Slack variables are defined in Eq. (7): (7) δi = 0 if ti −yi < ε ti −yi −ε otherwise , (7) (9) K xi, xj = exp −1 2σ 2 xi −xi2 , where ti is the ith target value of the model and yi is the ith predicted value of the model.h (9) The optimization problem in Eq. (8) is solved by its dual formulation more easily by introducing Lagrange multipliers, described as follows: where σ is the width of the RBF. After using a proper kernel function at last, the basic equation describing the modelling of the data is shown in Eq. 3.2 Support Vector Regression (10): (8) L = 1 2 w2 + C n i=1 δ+ i + δ− i − n i=1 η+ i δ+ i + η− i δ− i − n i=1 α+ i ε + δ+ i −yi + Y − n i=1 α− i ε + δ− i + yi −Y , (10) f x, α+ i , α− i = n i α+ i −α− i K xixj + b. (10) (8) where L is the Lagrangian function; η+ i , η− i , α+ i , and α− i are positive Lagrangian multipliers. Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 9 of 22 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 The biggest challenge in this algorithm is to find the optimum C, ε , and σ . The accuracy of an SVR closely depends on these values. In this study, the position of a salp and a grasshopper and chromosome of an individ- ual has three parts. The first part is dedicated to the C parameter, the second part is for the ε , and the third part is allocated to the σ. Each neuron in the hidden layers receives weighted inputs from the previous layer’s nodes and after summing them enter them in the activation function. The neurons in the output layer do the same without activation func- tion, and input layer nodes just receive input parameters from data records. An example of an ANN with one hid- den layer, two inputs, three nodes in the hidden layer and one output is illustrated in Fig. 7. 3.4 Optimization Algorithms Artificial Neural Network (ANN) is developed inspired by the human brain. If an ANN has enough inputs to learn, it can solve new problems. Multi-layer feed-for- ward back-propagation perceptron (MLFFBPP) is a kind of ANN in which there are an input layer and an output layer. Between these layers, there can be one or more lay- ers called hidden layers (Kandiri & Fotouhi, 2021; Shakor & Pimplikar, 2011). There are several neurons (nodes) in each layer connected to the next layer’s nodes with weighted links (Farooq et al., 2020; Lizarazo-Marriaga et al., 2020). In MLFFBPP, the flow of the information is from the input to the output layer. Then, weights of the network are modified in the back-propagation phase (Cybenko, 1989). ANNs use a learning algorithm to mod- ify their weights such as Bayesian regularization and Lev- enberg–Marquardt, which is used in this study because of its better performance (Golafshani & Behnood, 2018). Moreover, each node in the hidden layer includes an acti- vation function such as tangent sigmoid and hyperbolic tangent sigmoid that is used in the current paper. Accurate mathematical methods cannot solve optimiza- tion problems. Hence, to find the optimum answer pos- sible at a convenient time, heuristic and metaheuristic algorithms are used. The most popular algorithms are the ones that are developed inspired by nature (Behnood & Golafshani, 2018; Kandiri et al., 2020). Most of these algorithms consist of two parts: exploration and exploita- tion. The possible solutions that are far from each other are investigated in the exploration phase, then, in the exploitation phase, the close possible solutions are stud- ied. In fact, algorithms need the exploration phase to avoid local optimums. GA, SSA, and GOA are three of them. 3.4.1 Genetic Algorithm A genetic algorithm is a metaheuristic algorithm to solve optimization problems proposed by John Holland (1992). This nature-inspired algorithm uses Darwin’s evolution- ary theory. GA saves the data set in genes and each pat- tern of the dataset is recorded in an individual’s gene. Fig. 7 An illustrative of ANN. Page 10 of 22 Page 10 of 22 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 As mentioned before, every metaheuristic algorithm includes the exploration and exploitation phase, and GA uses mutation and crossover for these purposes, respec- tively. A number of individuals equal to the number of the initial population are going to survive based on their fitness function. In other words, the algorithm calculates the fitness function for each individual, and individu- als who are fitter have a better chance to survive. In this study, a roulette wheel is used to choose the survivors. This algorithm has been used to train an ANN in a num- ber of previous studies (Chandwani et al., 2015; Sahoo & Mahapatra, 2018; Shahnewaz & Alam, 2020; Shahnewaz et al., 2020; Yan et al., 2017; Yuan et al., 2014). stands at the front of the chain, and the rest of the group are followers. The exploration phase is the leader respon- sibility and the exploitation phase is handled by the followers.hi The position of each salp is defined in an n-dimension search space where n is the number of decision variables in the optimization problem. The following equation updates the leader position (Eqs. 11–13): P1 r = FPj + c1((UBr −LBr)c + LBr) c3 ≥0 FPj −c1((UBr −LBr)c2 + LBr) c3 < 0, (11) where LBr is the lower bound in the rth dimension, UBr is the upper bound in the rth dimension, FPj is the position of the food, Pi r is rth dimension of the leader position, c1 balances exploration and exploitation phases; c2 and c3 are random numbers in [0,1]. c1 is calculated as follows: 3.4.2 Salp Swarm Algorithmi Salps, which look like jellyfishes, belong to the Salpidae group, with a body shape like a transparent barrel (Mir- jalili et al., 2017). To coordinate rapidly for finding food, they usually create a chain. SSA is inspired by salps’ swarm intelligence. There is a leader in the chain that (12) c1 = 2e−( 4t T ) 2 , (12) Fig. 8 Example of the pseudocode using SSA. Fig. 8 Example of the pseudocode using SSA. Fig. 8 Page 11 of 22 Page 11 of 22 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 (17) Gi = −g eg, where T is the maximum number of iterations and t rep- resents the current iteration. The position of the follow- ers are calculated as follows: (17) (18) (18) Wi = u ew, (13) Pi r = 1 2 Pi r + Pi−1 r , (13) where g and eg are the constant of gravity and a unity vector towards the earth’s centre, respectively, and u and ew are the constant of gravity drift and a unity vector in the wind’s direction, respectively. A modified version of the is represented as follows (Eq. 19): where Pi r is position of the ith salp in the rth dimension. Now, it is possible to simulate the salp swarm. Fig. 8 illus- trates the pseudocode of SSA. This algorithm has been used to train an ANN in a number of previous studies (Kandiri & Fotouhi, 2021; Kandiri et al., 2020; Kang et al., 2019). (19) Xd i = m        N j = 1 j = i mubd −lbd 2 s xd j −xd i xj −xi dij        + Td, 3.5 Proposed Modelshi This section defines that how the proposed models are developed and how the optimization algorithms are com- bined by ANN and SVR. (15) Sr = n m = 1 m = r s(drm) drm, (15) 3.4.3 Grasshopper Optimization Algorithm (19)  j  (19) Although grasshoppers are observed individually in nature, they live in a huge swarm, and this behaviour is found in both nymph and adulthood (Rogers et al., 2003; Simpson et al., 1999). In their adulthood phase, they move in the long-range while their steps are small in their nymph phase. In fact, the big steps are for exploration and the small steps are for exploitation. Each grasshopper search agents have a position ( Xj ) made of n-dimensions, which is defined in Eqs. (14 and 15): where ubd and lb are the upper bound and the lower bound in the dth dimension, respectively, Td is the dth dimension of the target position, m is a decreasing coeffi- cient to shrink the comfort zone. In the first iteration, the rate of exploration is higher than that in the final itera- tions. Therefore, m should decrease as the algorithm get close to its end. The m parameter is calculated in Eq. (20): (20) m = mmax −ITmmax −mmin R , (20) i (14) Xj = c1Sj + c2Gj + c3Wj, (14) where IT is the number of maximum iterations and mmax and mmin are 1 and 0.00001, respectively. Fig. 9 shows the different steps of the GOA. where Sj , Gj , and Wj are social interaction, gravity force, and wind advection on the jth search agent. c1 , c2 , and c3 are random numbers between zero and one to make random behaviour. The following equation discusses the social interaction ( Sr): 4.2 Comparison of the Models’ Performances p After running the models, three ANNs and two SVRs are made. The architecture of ANNGOA, ANNSSA, and ANNGA are 9–7–4–1, 9–15–1, and 9–13–10–5–1, respec- tively. The weighted links and biases of these ANNs are rep- resented in Appendix 2. In other words, ANNSSA has the simplest architecture with 15 nodes in its only hidden layer, ANNGOA has the second simplest architecture with seven and five nodes in its first and second hidden layer, respec- tively, and ANNGA has the most complex one with three hidden layers including 13, 10, and 5 nodes in them, respec- tively. Moreover, the error limits, penalty coefficients, and the slack variables of SVRGOA are 5, 0.267, and 1 while these parameters of SVRSSA are 5, 0.237, and 1, respectively. These two SVRs are working almost the same. Fig. 11 dem- onstrates the errors of the models for the dataset. (21) RMSE = 1 Q Q j=1 (Mq −Vq)2. (21) 3.5.1 Modified ANNh The performance of an ANN is affected by its architec- ture; in fact, obtaining the optimum number of hidden layers and their nodes is the biggest challenge in build- ing a network. In the present paper, three different opti- mization methods are used to develop ANNs with the optimum architectures and reliable performances. The position of a search agent and the gene of an individual are divided into two parts. As shown in Fig. 10, the upper part is allocated to the existence of a hidden layer, and the lower part is allocated to the number of nodes in the hid- den layers. Each cell of the upper part can take a value of either 0 or 1. If the nth cell has the value of 1, the network includes the nth hidden layer, and if it has the value of 0, the networks do not include the mentioned hidden layer. Regarding the lower part, the mth cell indicates that the related hidden layer includes how many nodes. where drm is the distance between the rth and the mth grasshopper, calculated as drm = \|xm −xr\| , drm is a unit vector from the rth to the mth grasshopper as computed as drm = xm−xr drm , and s is a function to describe the social forces’ strength represented in Eq. (16): (16) s(r) = ia × e −r lc −e−r, (16) where ia and lc are the intensity of attraction and the attractive length scale, respectively. Based on the distance between two grasshoppers, they apply force on each other. This force could be absorption for far grasshoppers and repulsion for close grasshoppers. However, there is an exact value of distance, in which grasshoppers apply no force on each other, which is called comfort zone. Gi and Wi are calculated in Eqs. (17 and 18): Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 12 of 22 Kandiri et al. Int J Concr Struct Mater Fig. 9 Example of the pseudocode using GOA. Fig. 9 Example of the pseudocode using GOA. 4.1 Normalizationi In the first step, before entering the inputs into the model, it is needed to normalize them because of the difference in the scales of inputs. In this study, the following equation is used (Eq. 22): (22) an = 2(a −amin) (amax −amin) −1, (22) where a is an input value, amin , amax , and an are mini- mum, maximum, and normal values of the a, respectively. These three models have almost the same process as their original algorithms, but instead of a fitness function they use and ANN with the obtained architecture to calculate the fit- ness of each individual or search agent, and their errors are computed by root mean square error (RMSE) (Eq. 21): 4 Results and Discussion Fig. 10 The gene of and individual or the position of a search agent to optimize the ANN’s architecture. Fig. 10 The gene of and individual or the position of a search agent to optimize the ANN’s architecture. 3.5.2 Modified SVR Finding the best values for the penalty coefficient (C), the error limit ( ε ), and the slack variable ( δ ) are really important in the SVR method; in fact, the performance of an SVR is dependent on those parameters. To address this challenge in this paper, the gene of an individual and the position of a search agent are made with three cells. Each cell is allocated to one of the parameters. Therefore, the optimization algo- rithms can optimize the SVR performance and reduce its error as much as possible. The method that used in this sec- tion is similar to the method that used in Sect. 3.4.1. In this study, to compare the performances of the proposed models, in addition to RMSE, mean abso- lute percentage error (MAPE—Eq. 23), correlation Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 13 of 22 Fig. 11 Models’ errors (experimental values – predicted values). ental values – predicted values) Fig. 11 Models’ errors (experimental values – predicted values). Fig. 11 Models’ errors (experimental values – predicted values). coefficient (R—Eq. 24), mean absolute error (MAE— Eq. 25), scatter index (SI—Eq. 26), and mean absolute bias error (MBE—Eq. 27) are used: (25) MAE = 1 Q Q i=1 Mq −Vq , coefficient (R—Eq. 24), mean absolute error (MAE— Eq. 25), scatter index (SI—Eq. 26), and mean absolute bias error (MBE—Eq. 27) are used: (25) MAE = 1 Q Q i=1 Mq −Vq , (25) (23) MAPE = 100 Q Q i=1 Vq −Mq Vq , (26) SI = RMSE/ ¯M, SI = RMSE/ ¯M, (26) (23) (27) MBE = 1 Q Q i=1 Vq −Mq , (27) (24) R = Q Q i=1 MqVq  Q Q i=1 M2q − Q i=1 Mq 2   Q Q i=1 V 2q − Q i=1 Vq 2  , where M is the mean value of measured results, and other parameters are explained in the previous section. MBE indicates that the model overestimates (MBE > 0) or underestimates (MBE < 0). SI determines that the where M is the mean value of measured results, and other parameters are explained in the previous section. MBE indicates that the model overestimates (MBE > 0) or underestimates (MBE < 0). SI determines that the Kandiri et al. 3.5.2 Modified SVR Int J Concr Struct Mater (2022) 16:64 Page 14 of 22 performance of the model is “excellent” (0 ≤ SI < 0.1), “good” (0.1 ≤ SI < 0.2), “fair” (0.2 ≤ SI < 0.3), or “poor” (0.3 ≤ SI). Table 2 represents these indicators for the models. coefficient of 0.98. Fig. 12 illustrates the predicted values against the experimental values for the models in which it can be seen that the scatter around the baseline for all mod- els is almost the same. Furthermore, Fig. 13 compares the RMSE, MAE, and MAPE of the models in a radar chart in which, it can be seen that SVRSSA has the lowest RMSE, MAE, and MAPE values among all models. According to Table 2, SVRSSA has the lowest MAE fol- lowed by ANNGA while SVRGOA is following that closely, and ANNGOA and ANNSSA have the second-highest and highest MAE, respectively. Based on MBE, ANNGOA over- estimates the output while other models underestimate that. According to RMSE, SVRGOA, SVRSSA, and ANNGOA, are the first to third-best models, respectively, so that their distances are so low, and finally, ANNSSA and ANNGA are the worst and the second-worst ones, respectively. With regard to MAPE, SVRSSA has the best performance by far, SVRGOA has the second-best performance followed by ANNGA closely, and ANNGOA and ANNSSA are the fourth and fifth models. SI indicates that all of the models have fair performances. Finally, all models have a correlation 5 Conclusion Lateral pressure exertion from fresh concrete on the formwork panel creates uncertainty for industries while casting concrete. This is due to high pressure, particularly when the casting rate increases. Therefore, this study col- lected the various samples around the world to analyse and train in data learning. These machine learning appli- cations would be useful to investigate and predict the lat- eral pressure of concrete before casting and pouring into the formwork. So briefly the outcomes of this investiga- tion can be listed as follows: Fig. 13 A radar chart for comparing RMSE, MAE, and MAPE of the models. where Co is the initial pressure and time-dependent vari- able, and a is used to fit the function of pressure decay, while α is a time-dependent variable used to fit the func- tion to the pressure decay. Therefore, according to this formula most changes are in the density, casting rate and initial pressure. In addition, if the results in Fig. 8 are checked, most of the results are located between 50 to 70 kPa. This indicated that most of the height of the formwork is not more than 3 m cast at the same. How- ever, there are still some values that can be seen as over 100 kPa. • Following ACI 347-04 Guide to Formwork for Con- crete, it can be said that the lateral pressure exerted concrete in the real-world application did not record a value above 200 kPa. However, this mostly depends on the height of casting, casting rate, the con- stant value of gravity and the density of the materi- als; therefore, it could be expected to increase with increasing these parameters. • Generally, SVR-based models have better perfor- mances compared to ANN-based models, although all models have the same correlation coefficient approximately. Another study by Ovarlez and Roussel (2006) expressed the lateral pressure for rectangular and circular formwork can be obtained using Eqs. (29) and (30), respectively: • Based on MAE, MBE, and MAPE, SVRSSA is the most accurate model followed by SVRGOA closely. Nevertheless, SVRGOA has lower RMSE compared to SVRSSA. (29) Pmax = K2 ρgH −(H −e)2Athix LR , (30) Pmax = K2 ρgH −(H −e)2Athix rR , (29) • All machine-learning-based models have a high cor- relation coefficient, which indicates the great cor- relation between experimental and predicted lateral pressure. 4.3 Validation with Mathematical Modelling Mathematical modelling is always a decent method to predict the different characteristics of concrete. Hence, a study by Lange et al. (2008) used mathematical model- ling to predict the maximum lateral pressure of concrete while the casting, this is expressed in Eq. (28): (28) Ph = γ Rt C0 (at2 + 1)α , (28) Table 2 Models’ statistic indicators. Models MAE (MPa) MBE (MPa) RMSE (MPa) MAPE (%) SI R ANNGA 3.01 − 0.28 6.31 12.62 0.13 0.984 ANNSSA 4.08 − 0.32 7.24 18.22 0.15 0.979 ANNGOA 3.60 0.25 6.2 14.66 0.14 0.983 SVRSSA 2.60 − 0.06 6.16 8.20 0.13 0.985 SVRGOA 3.05 − 0.07 6.12 11.59 0.13 0.985 0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180 Predicted lateral pressure Experimental lateral pressure ANNGA ANNSSA SVRGOA SVRSSA ANNGOA Fig. 12 The experimental values (lateral pressure from the experimental test) vs the predicted values (the outputs of the model) of each model. Table 2 Models’ statistic indicators. 0 20 40 60 80 100 120 140 160 180 0 20 40 60 80 100 120 140 160 180 Predicted lateral pressure Experimental lateral pressure ANNGA ANNSSA SVRGOA SVRSSA ANNGOA Fig. 12 The experimental values (lateral pressure from the experimental test) vs the predicted values (the outputs of the model) of each model. Predicted lateral pressure Fig. 12 The experimental values (lateral pressure from the experimental test) vs the predicted values (the outputs of t Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Kandiri et al. Int J Concr Struct Mater Page 15 of 22 Page 15 of 22 Fig. 13 A radar chart for comparing RMSE, MAE, and MAPE of the models. In machine learning, all five models (ANNSSA, ANNGOA, ANNGA, SVRSSA, SVRGOA) yield excel- lent result values for R (coefficient of correlation) which recorded approximately 0.98 value for all models. This outcome is identical to use any of them as a prediction model for measuring lateral pressure of fresh concrete on the panel of the formwork. Appendix 1 See Table 3 5 Conclusion Therefore, all of them can be used to esti- mate the lateral pressure of concrete. (30) where r is the formwork radius, K2 is the ratio of lateral to vertical pressure and Athix is a flocculation coefficient of concrete. Based on these formulae show there are no dif- ferences between shape geometry. Their equations show clearly the height of the formwork has a major influence in an increase or decrease the lateral pressure exertion on the formwork. Nevertheless, there are not many differ- ences in the shape of the formwork for casting; therefore, it could not also see obvious differences. As a comparison with machine learning, these equations could be quite matched with all five models (ANNSSA, ANNGOA, ANNGA, SVRSSA, SVRGOA). • Even ANNSSA which is the least accurate model has an acceptable performance with the RMSE value of 7.24 and MAE value of 4.08. Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 16 of 22 Table 3 A listed result of the research on exerted lateral pressure of concrete on the formwork (Gowripalan et al., 2021). No. 5 Conclusion Int J Concr Struct Mater (2022) 16:64 Page 17 of 22 Table 3 (continued) No. 5 Conclusion Cement Water (W/C) Coarse aggregates Fine aggregates Admixture agent (ml/100 kg) Total height Casting rate Maximum pressure (k 1 218 0.88 997 665 3.28 3.00 6.00 71.00 2 218 0.88 997 665 3.28 3.00 2.00 61.00 3 218 0.88 997 665 3.28 5.00 6.00 113.00 4 218 0.88 997 665 3.28 5.00 2.00 83.00 5 218 0.88 997 665 3.28 7.00 6.00 151.00 6 218 0.88 997 665 3.28 7.00 2.00 88.00 7 450 0.40 870 740 7.8 2.80 10.00 49.00 8 400 0.47 569 1152 0 4.15 2.70 72.00 9 400 0.47 569 1152 0 6.58 5.06 83.00 10 420 0.39 645 1044 0 3.75 3.63 45.00 11 420 0.39 645 1044 0 4.11 5.13 62.00 12 420 0.39 645 1044 0 5.51 6.44 88.00 13 420 0.39 645 1044 0 4.20 3.27 70.00 14 420 0.39 645 1044 0 5.52 5.09 100.00 15 420 0.39 645 1044 0 4.15 3.19 52.00 16 450 0.35 944 872 5.9 3.90 10.00 58.00 17 280 0.75 905 735 2.4 12.00 71.00 156.80 18 280 0.75 905 735 2.4 12.00 71.00 186.20 19 280 0.75 905 735 2.4 12.00 71.00 166.60 20 280 0.75 905 735 2.4 12.00 71.00 176.40 21 280 0.75 905 735 2.4 12.00 71.00 117.60 22 315 0.55 862 862 6.5 2.70 18.80 55.00 23 325 0.45 305 610 2.7 2.70 18.80 35.00 24 450 0.42 890 710 6.88 2.10 10.00 45.00 25 300 0.49 900 900 10 0.47 2.70 92.00 26 280 0.78 891 735 2.85 2.00 2.50 52.00 27 450 0.40 870 740 7.8 2.80 25.00 58.00 28 280 0.78 891 735 2.85 2.00 2.50 52.00 29 365 0.53 715 805 7.7 2.00 25.00 43.00 30 220 0.91 770 440 0 0.76 0.55 35.00 31 232 0.86 812 464 0 1.06 0.55 26.00 32 303 0.66 682 379 0 1.31 1.16 47.88 33 182 1.10 910 380 0 2.01 2.30 29.70 34 400 0.50 1600 800 0 0.82 0.52 25.00 35 400 0.50 1600 800 0 0.88 0.55 11.50 36 400 0.50 1600 800 0 0.58 0.55 7.00 37 400 0.50 1600 800 0 1.04 1.10 10.50 38 400 0.50 1600 800 0 1.22 2.23 8.50 39 400 0.50 1600 800 0 0.85 2.26 14.00 40 400 0.50 1600 800 0 0.85 2.26 8.50 41 400 0.50 1500 900 0 0.61 0.46 5.00 42 400 0.50 1500 900 0 0.76 0.82 6.50 43 400 0.50 1500 900 0 0.76 1.01 5.50 44 400 0.50 2400 1200 0 0.64 0.52 6.50 45 400 0.50 2400 1200 0 0.52 0.49 4.00 46 400 0.50 2400 1200 0 0.76 1.01 3.00 47 400 0.50 2400 1200 0 1.07 1.07 6.00 48 400 0.50 2400 1200 0 1.16 2.04 1.50 Kandiri et al. 5 Conclusion Int J Concr Struct Mater (2022) 16:64 Page 18 of 22 Table 3 (continued) No. Cement Water (W/C) Coarse aggregates Fine aggregates Admixture agent (ml/100 kg) Total height Casting rate Maximum pressure (kPa) 97 500 0.40 750 750 0 1.83 3.05 29.33 98 500 0.40 750 750 0 3.05 3.05 45.07 99 500 0.40 750 750 0 3.66 3.05 51.31 100 500 0.40 750 750 0 4.88 3.05 60.91 101 500 0.40 750 750 0 6.10 3.05 64.51 102 500 0.40 750 750 0 1.83 4.27 29.33 103 500 0.40 750 750 0 3.05 4.27 45.07 104 500 0.40 750 750 0 3.66 4.27 52.75 105 500 0.40 750 750 0 4.88 4.27 69.31 106 500 0.40 750 750 0 6.10 4.27 79.54 107 500 0.40 750 750 0 1.83 5.49 29.33 108 500 0.40 750 750 0 3.05 5.49 45.07 109 500 0.40 750 750 0 3.66 5.49 55.44 110 500 0.40 750 750 0 4.88 5.49 69.84 111 500 0.40 750 750 0 6.10 5.49 88.46 112 320 0.75 825 915 0 2.00 10.00 45.00 5 Conclusion Cement Water (W/C) Coarse aggregates Fine aggregates Admixture agent (ml/100 kg) Total height Casting rate Maximum pressure (k 49 400 0.50 2400 1200 0 1.52 2.16 10.00 50 400 0.50 2400 1200 0 2.68 2.74 2.50 51 400 0.50 2400 1200 0 1.55 4.36 3.00 52 396 0.48 693 950 0 2.00 1.00 23.74 53 396 0.48 693 950 0 2.00 1.00 21.88 54 396 0.48 693 950 0 2.00 1.00 25.11 55 396 0.48 693 950 0 2.00 1.00 21.15 56 396 0.48 693 950 0 2.00 1.00 25.96 57 396 0.48 693 950 0 2.00 1.00 22.81 58 396 0.48 693 950 0 2.00 1.00 26.97 59 396 0.48 693 950 0 2.00 1.00 26.92 60 396 0.48 693 950 0 2.00 1.00 30.98 61 396 0.48 693 950 0 2.00 1.00 30.98 62 420 0.50 1030 715 0 17.00 5.00 100.00 63 330 0.50 900 630 0 3.00 12.00 60.90 64 330 0.50 900 630 0 2.00 12.00 59.43 65 330 0.50 900 630 0 3.00 12.00 65.15 66 330 0.50 900 630 0 2.00 12.00 65.20 67 330 0.50 900 504 0 3.00 12.00 58.38 68 330 0.50 900 504 0 2.00 12.00 62.37 69 330 0.50 900 504 0 3.00 12.00 65.48 70 330 0.50 900 504 0 2.00 12.00 65.29 71 330 0.50 900 504 0 3.00 12.00 61.14 72 330 0.50 900 504 0 2.00 12.00 65.19 73 370 0.50 1047 758 2 3.00 1.29 27.00 74 370 0.50 1047 758 0 3.00 1.33 37.50 75 370 0.50 1047 758 0 3.00 0.93 41.50 76 370 0.50 1047 758 0 3.00 1.16 30.50 77 370 0.50 1047 758 0 3.00 0.45 27.50 78 370 0.50 1047 758 0 3.00 8.00 28.50 79 370 0.50 1047 758 0 3.00 1.45 38.20 80 370 0.50 1047 758 0 3.00 1.16 27.20 81 370 0.50 1047 758 0 3.00 1.97 24.80 82 370 0.50 1047 758 0 3.00 3.30 31.30 83 370 0.50 1047 758 0 3.00 6.38 33.00 84 370 0.50 1047 758 0 3.00 1.50 54.70 85 370 0.50 1047 758 0 3.00 2.36 52.90 86 370 0.50 1047 758 0 3.00 5.17 43.70 87 500 0.40 750 750 0 1.83 0.61 14.83 88 500 0.40 750 750 0 3.05 0.61 14.83 89 500 0.40 750 750 0 3.66 0.61 14.83 90 500 0.40 750 750 0 4.88 0.61 14.83 91 500 0.40 750 750 0 6.10 0.61 14.83 92 500 0.40 750 750 0 1.83 1.83 27.55 93 500 0.40 750 750 0 3.05 1.83 37.82 94 500 0.40 750 750 0 3.66 1.83 37.82 95 500 0.40 750 750 0 4.88 1.83 37.82 96 500 0.40 750 750 0 6.10 1.83 37.82 Kandiri et al. Biases and Weights of the ANNSSA Model Weights of links between input and the hidden layer =   0.94 0.44 0.32 1.32 −1.21 0.33 0.43 −0.47 −0.38 0.56 −1.10 0.25 0.44 −0.33 0.20 0.32 −0.42 0.03 −0.14 0.20 0.48 0.71 0.72 −0.40 0.49 0.62 0.02 −0.63 0.64 0.77 0.72 −0.13 0.03 0.58 0.64 −0.81 −0.20 0.94 0.62 −1.23 −1.21 −0.54 0.02 −0.70 1.91 0.41 −0.59 1.36 1.43 −1.09 0.48 −0.33 0.78 −0.06 −0.58 0.19 −0.18 0.14 1.16 0.97 −0.44 0.67 −0.07 0.53 −0.82 −0.79 0.03 −1.50 −0.66 −1.01 0.97 0.03 −1.11 −2.52 −0.037 −0.06 0.11 1.36 −0.84 −1.17 −0.56 −0.096 −0.95 −0.67 −0.84 −0.58 0.16 1.31 1.75 −0.78 −1.33 −0.36 −0.43 −0.41 0.27 −0.41 −1.15 0.61 −1.48 −0.31 0.02 0.30 1.25 1.75 0.16 −0.76 0.01 −0.71 0.70 0.4 0.22 −0.39 0.24 0.14 0.48 0.35 −1.41 −0.67 1.78 1.59 0.18 −0.14 0.30 0.63 −0.09 0.9 0.70 0.00 0.58 0.25 0.43 −0.50 2.30 0.65 −4.06   . Biases of the input layer = 0.83 1.22 −0.25 −0.01 −0.53 −0.21 1.27 0.43 −0.71 0.64 0.07 0.90 0.58 1.28 −1.32 . Weights of links between hidden and output layer = −0.28 −0.31 −0.67 0.86 −0.17 −0.78 0.44 0.87 0.48 −0.23 −0.82 −0.91 −1.22 1.18 −1.92 . eights of links between hidden and output layer = −0.28 −0.31 −0.67 0.86 −0.17 −0.78 0.44 0.87 0.48 −0.23 −0.82 −0.91 Biases of the hidden layer = −0.18 . Page 19 of 22 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Kandiri et al. Int J Concr Struct Mater Biases and Weights of the ANNGOA Model Weights of links between input and the ﬁrst hidden layer =   −0.22 0.06 −1.00 1.44 0.78 0.23 −3.02 1.15 0.67 2.37 −3.05 2.13 2.68 0.57 −0.90 −0.09 −0.48 1.68 0.45 0.70 −1.16 −0.78 0.32 −0.12 −1.93 2.40 0.79 −0.13 −0.61 −1.12 1.15 −1.32 0.26 −0.26 1.52 −1.95 −0.99 −2.58 0.64 −2.31 −0.56 0.62 0.29 −1.66 0.71 1.71 0.57 1.84 2.45 −1.73 1.81 −2.50 1.18 1.37 1.10 −1.03 −0.091 −0.31 −0.37 −5.13 −3.87 −0.12 2.03   . Weights of links between input and the ﬁrst hidden layer =   −0.22 0.06 −1.00 1.44 0.78 0.23 −3.02 1.15 0.67 2.37 −3.05 2.13 2.68 0.57 −0.90 −0.09 −0.48 1.68 0.45 0.70 −1.16 −0.78 0.32 −0.12 −1.93 2.40 0.79 −0.13 −0.61 −1.12 1.15 −1.32 0.26 −0.26 1.52 −1.95 −0.99 −2.58 0.64 −2.31 −0.56 0.62 0.29 −1.66 0.71 1.71 0.57 1.84 2.45 −1.73 1.81 −2.50 1.18 1.37 1.10 −1.03 −0.091 −0.31 −0.37 −5.13 −3.87 −0.12 2.03   . Biases of the input layer = 0.03 −0.22 −0.34 −0.55 −0.98 −1.07 0.09 . Biases of the input layer = 0.03 −0.22 −0.34 −0.55 −0.98 −1.07 0.09 . Weights of links between the ﬁrst and the last hidden layer =   −1.11 −0.66 −2.24 −0.78 2.39 −0.00 2.62 1.05 −0.36 1.21 2.38 0.68 1.57 −0.15 2.68 0.78 0.11 −1.65 −4.72 −1.34 −1.90 −0.59 1.45 −0.11 2.15 −0.63 0.93 0.54   . Biases of the ﬁrst hidden layer = −0.04 −1.24 0.55 −0.33 . Weights of links between the last hidden and output layer = −0.10 −1.14 −0.68 2.65 . Biases of the last hidden layer = [−0.20]. Biases of the last hidden layer = [−0.20]. Biases and Weights of the ANNGA Model Weights of links between the last hidden and output layer = 1.96 0.79 1.58 0.70 0.06 . Weights of links between the last hidden and output layer = 1.96 0.79 1.58 0.70 0.06 . Consent for publication Not applicable. Consent for publication Not applicable. Competing interests Competing interests The authors declare no competing interests. Authors’ information 1 School of Civil Engineering, University College Dublin, Belfield Dublin 4, Ireland. 2 Technical College of Engineering, Sulaimani Polytechnic University, Sulaymaniyah 46001, Iraq. 3 Civil Engineering Department, Tishk International University, Sulaymaniyah 46001, Iraq. 4 ICONSMAT, Sydney, NSW 2748, Australia. 5 Department of Highway and Bridge Engineering, Technical Engineering College, Erbil Polytechnic University, Erbil 44001, Iraq. 6 Department of Civil Engineering, College of Engineering, Nawroz University, Duhok 42001, Iraq. 7 CERIS, Civil Engineering, Architecture and Georesources Department, Insti- tuto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049‑001 Lis- bon, Portugal. 8 Structural Engineering Department, Structural Engineering and Construction Management, Future University in Egypt, New Cairo 11835, Egypt. Amirreza Kandiri, PhD Candidate, University College Dublin. Pshtiwan Shakor, Lecturer, Sulaimani Polytechnic University/ Tishk International University. Rawaz Kurda, Lecturer, Erbil Polytechnic University/ Nawroz University. Ahmed Farouk Deifalla, Professor, Future University. Funding No funding was received. Biases of the ﬁrst hidden layer = −0.56 0.00 −0.51 0.55 0.55 −0.26 −0.40 0.09 0.46 0.24 . Weights of links between the second and the last hidden layer =   0.24 −0.90 −0.97 −0.18 −0.68 0.78 −0.38 1.18 −0.18 0.85 1.02 −0.94 0.05 1.00 0.61 −0.88 −0.44 0.22 1.16 −0.43 0.31 −0.48 1.32 −0.36 −0.23 −1.07 −0.56 1.29 −0.69 −0.32 −0.16 0.62 −0.55 0.83 0.54 0.96 1.02 −0.98 −0.38 −0.26 0.61 0.22 0.77 −1.00 0.45 0.77 0.26 −0.93 0.46 −0.57   . Biases of the ﬁrst hidden layer = 0.33 0.95 0.70 0.54 0.07 . Acknowledgements h h ld l k The authors would like to thank the tremendous support from the Future University in Egypt, specifically Professor Ahmed Farouk Deifalla. Author contributions All authors contributed equally in this paper. PS and AK wrote the first draft of the paper. RK and AFD revised the paper completely with detailed comments and improvement. All authors read and approved the final manuscript. Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Biases and Weights of the ANNGA Model Weights of links between input and the ﬁrst hidden layer =   0.16 −0.56 −0.89 0.07 −0.30 0.17 −0.60 −1.08 0.81 −0.28 −0.83 −0.73 −0.022 −0.25 −0.28 −0.08 −0.70 −0.89 −0.02 −1.05 −0.91 1.13 −0.49 −0.99 −0.57 1.096 −0.31 0.80 1.05 −0.67 −1.16 0.40 0.69 0.55 0.35 0.94 0.02 −0.11 0.013 0.15 −0.10 −0.95 −0.43 −0.95 0.09 0.22 0.85 −0.99 0.17 0.72 −0.73 0.51 −1.22 −0.22 0.39 −0.86 −0.79 −0.41 −0.50 −0.50 −0.26 −0.84 −0.63 0.52 0.34 0.20 −0.72 −0.18 0.53 0.45 0.88 0.32 −0.45 −0.09 −0.96 −0.40 0.13 0.38 0.40 −0.01 0.76 1.00 0.45 −0.14 −0.79 −0.31 0.96 −0.99 −1.09 0.76 0.61 −1.11 0.01 0.35 0.57 1.06 −0.81 0.52 −1.05 −1.53 0.13 −0.79 1.06 −0.12 0.87 −0.63 −1.02 0.21 0.06 0.86 −0.27 −0.73 0.03 0.48 1.90 0.78 −1.47   . Biases of the input layer = −0.69 0.86 −0.96 0.25 −0.10 0.77 0.91 −0.20 0.50 0.12 −0.41 −0.41 0.39 . Biases of the input layer = −0.69 0.86 −0.96 0.25 −0.10 0.77 0.91 −0.20 0.50 0.12 −0.41 −0.41 0.39 . nput layer = −0.69 0.86 −0.96 0.25 −0.10 0.77 0.91 −0.20 0.50 0.12 −0.41 −0.41 0.39 . Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Kandiri et al. Int J Concr Struct Mater (2022) 16:64 Page 20 of 22 Weights of links between the ﬁrst and the second hidden layer =   1.07 0.51 0.73 −0.12 −0.72 0.71 −0.62 −0.37 0.40 −0.21 −0.96 1.14 −0.87 0.78 0.07 −0.29 0.08 −0.14 −0.48 0.16 −0.92 −0.8 0.27 0.66 −0.53 −0.57 −0.90 −0.43 0.71 0.05 0.72 −0.07 −0.59 −0.96 −0.85 0.02 0.17 −0.38 −1.07 −0.40 0.93 0.10 0.59 −1.09 0.89 0.38 0.92 0.15 −0.23 −0.42 0.38 −0.14 −0.80 0.51 0.30 0.89 −0.05 0.25 −0.75 −0.31 −0.48 −0.90 0.42 0.71 −0.38 0.82 −0.29 0.76 −0.65 0.54 0.53 −0.01 −0.41 0.36 0.08 −0.36 0.21 1.23 0.46 −0.14 −0.47 −0.41 −0.19 −0.65 −1.28 0.52 −0.44 0.99 −0.52 −1.10 0.058 −0.76 −0.05 0.16 0.35 0.018 0.74 −0.19 −0.10 −0.46 1.04 −0.58 0.40 0.27 1.27 1.14 0.48 0.72 −0.28 −0.17 0.86 0.63 0.51 −0.19 −0.71 0.42 0.24 0.35 0.54 −0.11 −1.11 −0.23 −0.14 0.25 −1.08 −1.27 −0.23 0.69 0.01 0.81   . Biases of the ﬁrst hidden layer = −0.56 0.00 −0.51 0.55 0.55 −0.26 −0.40 0.09 0.46 0.24 . 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https://openalex.org/W3042090573	http://revistas.unisimon.edu.co/index.php/innovacioning/article/download/3857/4597, https://dialnet.unirioja.es/descarga/articulo/7799062.pdf	es		Tecnologías, procesos y problemática ambiental en la Minería de arcilla	Investigación e innovación en ingenierías	2,020	cc-by	10,313	Tecnologías, procesos y problemática ambiental en la Minería de arcilla Technologies, processes and environmental problems in clay mining Danny Daniel López Juvinao , Luis Miguel Torres Ustate , Fabio Orlando Moya Camacho Universidad de La Guajira, Colombia Open Access Recibido: 28 de enero de 2020 Aceptado: 16 de marzo de 2020 Publicado: 9 julio de 2020 Correspondencia: dlopezj@uniguajira.edu.co DOI: https://doi.org/10.17081/invinno.8.2 .3857 Resumen Objetivo: Realizar un estudio sobre las tecnologías, problemática ambiental y procesos ambientales en empresas mineras que explotan arcilla en La Guajira (Colombia). Metodología: La investigación se fundamentó en el paradigma epistemológico postpositivista, de carácter descriptiva y sus fuentes provienen de revisión documental y observación directa, con diseño de campo, no experimental y de corte transeccional. La población estuvo conformada por cinco (5) empresas mineras: Camarones, San pedrito, El Confuso, Loma Fresca y La Junta, dedicadas a la extracción de arcilla y comercialización de ladrillos en La Guajira. Para recolectar la información, se aplicó la matriz de Leopold y un cuestionario estructurado por 24 ítems, aplicando la validez y confiabilidad mediante el criterio de expertos. Resultados: Se encontró que las tecnologías empleadas en las minas son de tipo artesanal y rudimentarias, evidenciándose un patrón de externalidades ambientales ocasionadas al medio físico, biológico y socioeconómico. Así mismo, se halló una desarticulación entre la ley general ambiental, el código minero y los planes de ordenamiento territoriales donde se localizan las minas, siendo esta una de las causas de conflictos ambientales y territoriales en las minas. Conclusiones: En definitiva, se identificaron cinco (5) impactos al medio físico y biológico y cuatro (4) impactos al medio socioeconómico, para un total de nueve (9) externalidades ambientales que afectan la zona de influencia de cada una de las minas; en efecto, se sugiere tecnificar los procesos mineros, mediante tecnologías limpias e inserción de procesos ambientales que ayuden a la gestión socioambiental de las empresas mineras. Palabras claves: Tecnologías, problemática ambiental, procesos ambientales, empresas mineras, minería, arcilla, ladrilleras. Abstract Objective: To conduct a study on technologies, environmental problems, and environmental processes in mining companies that exploit clay in the Guajira region (Colombia). Methodology: The research was based on the post-positivist epistemological paradigm with a descriptive approach, and its sources come from documentary review and direct observation, with field, non-experimental, and cross-sectional designs. The population comprised five mining companies, i.e., Camarones, San Pedrito, El Confuso, Loma Fresca, and La Junta, dedicated to the extraction of clay and marketing of bricks in the Guajira region. To collect the information, the Leopold matrix and a questionnaire structured by 24 items were applied, implementing the validity and reliability through criteria set by experts. Results: It was found that the technologies used in the mines are handmade and rudimentary, showing a pattern of environmental externalities that affect the physical, biological, and socioeconomic environments. Likewise, a disarticulation was found between the general environmental law, mining code, and land use planning at the location of the mines, this being one of the causes of environmental and territorial conflicts in the mines. Conclusions: In short, five impacts on the physical and biological environments and four impacts on the socioeconomic environment were identified, for a total of nine environmental externalities affecting the zone of influence of each of the mines; in particular, it is suggested to technically improve mining processes using clean technologies and the insertion of environmental processes to facilitate the socioenvironmental management of the mining companies. Keywords: Technologies, environmental problems, environmental processes, mining companies, mining, clay, brick companies. © Copyright: Investigación e Innovación en Ingenierías Como citar (IEEE): D.D. López - Juvinao., L.M. Torres - Ustate., y F.O. Moya - Camacho, “Tecnologías, procesos y problemática ambiental en la Minería de arcilla”, Investigación e Innovación en Ingenierías, vol. 8, n°. 2, 2020. DOI: https://doi.org/10.17081/invinno.8.2.3857 Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020. DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Introducción Actualmente, las dinámicas globales siguen un modelo lineal capitalista de producción a gran escala, que se basa en el consumo y dilapidación de los recursos naturales no renovables. Este modelo, a su vez, está soportado en el necesario consumo de la oferta ambiental que ha crecido exponencialmente por el auge de la tecnología, el crecimiento demográfico y las necesidades no justificadas que crea el sistema industrial [1, 2]. Esto indica que la mayoría, por no decir que todos los productos, requieren del uso de los recursos naturales para su elaboración, y para lograr satisfacer la demanda se ha recurrido a la explotación de múltiples territorios provocando la exacerbación sin precedentes del ambiente [3]. Dentro de este marco, la relación entre los procesos de crecimiento basados en la producción industrializada de bienes y los servicios que consumen energía originada en la quema de combustibles fósiles han generado un calentamiento global [1, 4], que según evidencias científicas internacionales, este indica un cambio en los fenómenos meteorológicos extremos en el planeta, llamado cambio climático (CC), que actualmente constituye un tema relevante tanto en la agenda política como en la opinión pública [5] y ha sido planteado por algunos autores como la problemática ambiental del siglo XXI [6, 7, 8], en relación con estas implicaciones, recientemente los países adoptaron el Acuerdo de París en la COP21 en París y con ello los Objetivos de Desarrollo Sostenible (ODS) como respuesta a fortalecer la respuesta global a la amenaza del CC [9]. A pesar de este esfuerzo, los conflictos socioambientales son cada vez más frecuentes en América Latina debido al desarrollo de políticas públicas extractivistas [10, 11], en las que incluso gobiernos progresistas han mostrado retrocesos en el terreno ambiental y social por no contar con una sostenibilidad corporativa dentro del sector minero [12]. Este escenario de conflictividad alcanza niveles mayores en países como el de Colombia, donde el modelo extractivista se ha instaurado como una de las principales actividades económicas, sin embargo, las tecnologías, los procesos ambientales, la pobre regulación, el escaso control de las problemáticas ambientales, la falta de presencia del Estado y la protección de intereses particulares producidas por estas actividades extractivas, han puesto en evidencia complejos conflictos de carácter socio ambiental que repercuten profundamente en las dinámicas poblacionales y que comienzan a ser motivo de preocupación debido a los efectos que se entrevén en las condiciones de vida, salud y el tejido social propio de las poblaciones aledañas a las zonas de explotación de minerales [8, 13, 14]. Sumado a esto, existen cifras económicas a nivel mundial que estiman que más de 30 millones de personas se dedican a la exploración y explotación de minerales de manera informal, utilizando tecnologías y técnicas artesanales Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho rudimentarias sometidas a prácticas inseguras y sin involucrar procesos ambientales adecuados que impidan la generación de problemáticas ambientales [15]. Estas cifras en Colombia se estiman en más de 202 mil empleos generados por el sector de minas y canteras [16, 17], convirtiéndose en un sector importante para la economía del país, ya que aporta el 56% del PIB y representa 13.835 puestos de trabajo de forma directa, donde solo en la extracción de carbón genera ~92% de estos empleos [18]. Se observa también, que en Colombia, La Ley 685 de 2001 (Código de Minas) prácticamente coloca al Estado como un observador en cuanto a la actividad minera, incentivando la inversión privada, lo cual se ratifica en los Planes de Desarrollo Minero desde el año 2002 hasta la actualidad [19, 20], situación que afecta al sector ambiental, ya que hasta hace poco menos de 10 años se entregaron cientos de títulos mineros para la explotación de áreas protegidas, lo que ha ocasionado enormes problemáticas ambientales y sociales, esto debido a que ni el Ministerio de Ambiente y Desarrollo Sostenible, ni las corporaciones autónomas regionales, han definido en su totalidad el deber ser de los procesos ambientales y el régimen de uso de las zonas que les corresponden [19]. Lo anteriormente expuesto, ha llevado a que en La Guajira las minas de arcilla o también llamadas ladrilleras actúen de manera informal, donde tradicionalmente se utiliza una tecnología rudimentaria, consistente en herramientas manuales que incrementan el esfuerzo físico en la ejecución de las tareas, evidenciando que las problemáticas ambientales causadas por parte de la extracción y procesamiento de la arcilla son de consideración y se debe principalmente a que no se llevan a cabo procesos ambientales en este tipo de minería, que además de causar una problemática que influye en lo ambiental, también afecta el componente social [21, 22]. Atendiendo a estas consideraciones, en La Guajira se está llevando a cabo un programa de formalización, que en efecto se encuentra en proceso de ejecución cuyo objetivo es lograr la caracterización del 100% de las unidades mineras, donde llama la atención que en las empresas mineras dedicadas a la explotación de arcilla, ya se viene trabajando sobre la formalización y regularización de las canteras que se encuentran en municipios como: Fonseca, Riohacha, Distracción, El Molino, Barranca y San Juan del Cesar [18]. De esta manera, este sería el primer paso para implementar tecnologías acordes a los procesos mineros, teniendo en cuenta la problemática ambiental y los procesos ambientales que se lleven a cabo en las minas de arcillas y otros minerales. En el marco de esta dinámica nacional y regional, el presente trabajo explora los vínculos entre la tecnología, los procesos ambientales y la problemática ambiental en las minas de arcillas acentuadas en el departamento de La Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Guajira, específicamente en las empresas mineras de Loma Fresca y La Junta en San Juan, El Confuso en Fonseca, San Pedrito en Barrancas y Camarones en el Distrito Especial, Turístico y Cultural de Riohacha, donde la presencia de las ladrilleras han causado conflictos entre las entidades gubernamentales (discrepancia entre el Código de Minas, Ley general ambiental y los Plan de Ordenamiento Territorial), las canteras y las comunidades ubicadas a los alrededores de la explotación, a causa de los efectos producidos tanto en el ambiente como en la salud de la población. En este orden de ideas, se hace improcedente garantizar un equilibrio entre el desarrollo de las minas y el consumo de la oferta ambiental, si no existe un adecuado control gubernamental para llevar a cabo los procesos ambientales, principalmente en las minas dedicadas a la extracción de arcilla. En La Guajira, esta problemática ambiental ha provocado la degradación de muchos ecosistemas, al no efectuarse ningún tipo de estrategias para prevenir, mitigar, controlar y/o compensar las externalidades ambientales que las ladrilleras causan en lo social y lo ambiental. Sobre la base de las ideas expuestas, en el ámbito teórico, esta investigación es importante porque cubre muchos interrogantes, aportando al conocimiento existente sobre las tecnologías, procesos ambientales y problemática ambiental en las empresas mineras dedicadas a la extracción de arcilla en La Guajira, teniendo en cuenta que es una minería a cielo abierto e identificando los impactos socio-ambientales generados. Por ello, en el modelo de esta investigación se tiene en cuenta la planificación de los recursos naturales en las diferentes etapas de ejecución de las actividades mineras, definiendo que las estrategias sean viables técnica, ambiental, social y económicamente, para proteger el medio ambiente y mejorar la calidad de vida de los trabajadores y comunidades afectadas. En la parte práctica, esta investigación es substancial ya que conocer las tecnologías y los procesos ambientales, resuelve el problema socioambiental en las ladrilleras de La Guajira contribuyendo a una minería organizada y responsable con el medio ambiente, donde se facilite la obtención de licencias y permisos ambientales; en pocas palabras, se disminuye el número de empresas mineras dedicadas a la extracción de arcillas asentadas en el departamento de La Guajira. Metodológicamente, esta investigación es importante porque lleva a que las diferentes empresas mineras dedicadas a la extracción de arcilla en La Guajira, tengan en cuenta como desarrollar un modelo para implementar tecnologías y procesos limpios en sus prácticas mineras, teniendo en cuenta las problemáticas ambientales que se presentan en la localización de sus empresas y su afectación socio-ambiental en el área de influencia, además de la maquinaria, su producción y procesos ambientales, con el fin de promover una minería responsable. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho Metodología La investigación se fundamentó en el paradigma epistemológico post positivista, de tipo adaptativa y carácter descriptivo, con diseño de campo, no experimental y de corte transeccional [23, 24], cuyas fuentes provienen de revisión documental y observación directa [25]. En el presente estudio no se trabajó con muestras, sino con la población total, cuya población fue de carácter finita, donde hubo accesibilidad a las empresas mineras dedicadas a la minería de arcilla, representada en cinco (5) minas (Camarones S. A, San pedrito S. A, El Confuso S. A, Loma Fresca S. A y La Junta S. A), dedicadas a la extracción de arcilla y comercialización de ladrillos (Ladrilleras) en La Guajira [21, 22]. La investigación se realizó mediante la observación en campo a las minas de arcilla en La Guajira, sobre las siguientes dimensiones junto a sus indicadores y/o criterios de evaluación, como se muestra en la Tabla 1. Tabla 1. Dimensiones, indicadores y criterios de evaluación en las minas de arcilla de La Guajira Dimensiones Criterio de evaluación Se identificaron las tecnologías utilizadas en los procesos mineros Tecnología para la explotación de arcilla. Para proponer procesos ambientales eficaces se establecieron una Procesos ambientales serie de indicadores como: la oferta ambiental, valoración ambiental, viabilidad ambiental y conflictos ambientales, que se presentan actualmente en las empresas mineras objeto de estudio. Se realizó una evaluación de impacto ambiental para determinar qué tipo de actividades y componentes ambientales requieren mayor atención en este tipo de minería, además de profundizar en aspectos de interés como lo son: la cultura ambiental, carga contaminante, Problemática ambiental efectos a la salud, problemas de medio físico de las empresas mineras objeto de estudio. Es importante resaltar que en este documento no se profundizo en los impactos sociales que el proyecto minero representa, sin embargo, se evalúa como punto de referencia para nuevas investigaciones. Fuente: Elaboración propia Además de la observación en campo se contó con información por parte de los trabajadores de las minas en las cuales se realiza la explotación y procesamiento de la arcilla en La Guajira (ver Tabla 2). Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Tabla 2. Empresas mineras que explotan arcilla en L a Guajira y número de trabajadores vinculados Municipio Empresas mineras Número de trabajadores Barrancas San pedrito S. A 20 Fonseca El confuso S. A 25 San Juan del Cesar Loma Fresca S.A y La Junta S. A 30 Riohacha Camarones S. A 25 Total 100 Fuente: Elaboración propia La técnica de recolección utilizada en esta investigación fue una matriz de Leopold, como herramienta para un seguimiento continuo en la evaluación de impacto ambiental en las empresas mineras de arcilla en La Guajira [26] y un cuestionario [23], para analizar la situación ambiental en las minas, con un modelo de pregunta y respuesta abierta, que permitieron arrojar información para llegar a obtener discusiones y resultados, conclusiones finales, así como recomendaciones e investigaciones futuras en las empresas mineras dedicadas a la explotación de arcilla en La Guajira. De acuerdo con la matriz Leopold, esta estuvo compuesta por factores ambientales receptores de impactos (FARI) y acciones de las empresas mineras susceptibles de producir impactos (ASPI) en la minería de arcilla de La Guajira. Donde para arrojar la matriz final, se sintetizo la información recolectada en todas las minas objetos de estudio y se evaluó de manera integral los problemas socioambientales que causan, dando resultado una matriz completa que abarca todos los FARI y las ASPI a lo largo de la explotación de arcilla en La Guajira. En la tabla 2, se muestran los criterios de juicio que se tuvieron en cuenta para la evaluación de impacto ambiental (EIA) en las empresas mineras que explotan arcilla. Donde, se categorizaron las externalidades ambientales según la magnitud e importancia, en el que la magnitud se midió en un rango de (-10 hasta +10) y el rango de Importancia se midió de (+1 hasta +10), los cuales fueron evaluados según el criterio de los autores a partir de [27, 28]. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho Tabla 3. Categorización de externalidades ambientales para evaluac ión de impactos ambientales en las minas de arcilla de La Guajira Magnitud Importancia Alteración Grado de destrucción Baja Media Calificación Persistencia Extensión Calificación (+) 1 Fugaz Puntual 1 2 Temporal Puntual 2 3 3 Permanente Puntual 3 Medio 4 4 Fugaz Parcial 4 Medio 5 5 Temporal Parcial 5 Alta Medio 6 6 Permanente Parcial 6 Baja Alto 7 7 Fugaz Extrema 7 Media Alto 8 8 Temporal Extrema 8 Alta Alto 9 9 Permanente Extrema 9 Alta Muy alto 10 10 Permanente Total 10 (-) (+) Mínimo 1 Mínimo 2 Alta Mínimo Baja Media Fuente: Elaboración propia partir de [ 27, 28] Por otra parte, el cuestionario se estructuró por 24 ítems, que consto de una serie de tres (3) preguntas abiertas o ítems por cada uno de los indicadores, aplicada a cada una de las empresas mineras que explotan arcilla en La Guajira. Así mismo, con el objeto de efectuar la validez de esta investigación se tuvo en cuenta el criterio de consulta técnica a expertos en las áreas de metodología de la investigación, en ciencias ambientales y minería en La Guajira, evaluando que el instrumento fuese pertinente en relación a su alcance, contenido, efectividad, coherencia, redacción, entre otros. Atendiendo a estas consideraciones, la confiabilidad de esta investigación no es medida, por cuanto este estudio está sujeto a criterios propios como investigadores tanto en la observación en campo como para la selección de material documental; en su virtud, el instrumento fue evaluado por expertos, quienes determinaron su confiabilidad al momento de recolectar información de interés. Resultados y discusión Tecnologías De acuerdo con [29], en el departamento de La Guajira no se implementan las tecnologías acordes a los procesos mineros y actividades que se llevan a cabo, ya que las empresas no toman en cuenta y no integran la legislación ambiental o simplemente se desvinculan del componente socioambiental porque les representa elevados costos. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Es así, como actualmente las tecnologías utilizadas en los procesos mineros de las empresas mineras objeto de estudio dedicadas a la explotación de arcilla en La Guajira, son de tipo artesanal y/o rudimentarias que consisten en herramientas manuales que incrementan el esfuerzo físico de los trabajadores en la ejecución de las diferentes actividades (ver Tabla 4). Situación que repercute en que exista una baja capacidad de producción en las empresas mineras, causan problemas ambientales y afecta los procesos ambientales y legales en las empresas mineras. Tabla 4. Tecnologías utilizadas en los procesos mineros de explotación de arcilla en La Guajira Proceso minero Uso Paladragas Prospección y azadón Se utilizan en trabajos preliminares, para hacer pequeñas excavaciones que permita observar si el material arcilloso puede ser explotado. Palas, machetes y Se utilizan para la remoción de la capa vegetal que cubre el Descapote Extracción Tecnología motosierra de Azadón, arcilla material arcilloso. palas y paladragas Azadón, palas, baldes y amasado Mezclado manual Se utilizan para sacar la arcilla del subsuelo. (con las Se utilizan para revolver y homogenizar el material arcilloso. manos) Molde de madera o Se utiliza para moldear la mezcla de arcilla y darles una forma Moldeado de hierro Secado de los ladrillos Cocción de ladrillos rectangular a los ladrillos. Se dejan los ladrillos en exposición a los rayos solares al aire libre, Radiación solar para que los ladrillos pierdan humedad. Los ladrillos se utilizan para la construcción de hornos Ladrillos y leña artesanales y la leña para la quema de ladrillos. Se utilizan las carretillas para trasladar los ladrillos dentro de la Despacho y Traslado de los ladrillos Carretillas y plásticos mina y los plásticos para cubrir los ladrillos almacenados luego de ser quemados y haber recuperado una temperatura ambiente, y quedar listos para su posterior traslado al destino final que se da mediante camiones. Fuente: Elaboración Propia Por esta razón, y en referencia a esta problemática se presume que a nivel mundial existen paquetes tecnológicos transferidos de otros países, en los que la normatividad ambiental es rigurosa y obliga a un mejoramiento de las tecnologías convencionales a las de producción limpia y además exige a las empresas que dentro de estos paquetes tecnológicos se incluya un paquete ambiental. Dentro de este marco, se hace importante abrir campo a la transferencia tecnológicas que funcione como un plus de innovación en las Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho empresas mineras, aumentando su productividad y las ayude a mantenerse en el mercado actual, seleccionando, adquiriendo, adaptándose y poniendo en marcha una tecnología obtenida conforme a las necesidades propias de las actividades mineras[29]. Así pues, la recomendación a las minas dedicadas a la explotación de arcilla en La Guajira es la implementación de nuevas tecnologías que permitan el desarrollo de sus empresas y eviten daños y/o problemáticas ambientales. Procesos ambientales De acuerdo con la Norma Técnica Colombiana NTC-ISO 14001 [30], los procesos ambientales son las instrucciones, paso a paso, que realizados de una forma adecuada permite controlar el sistema de gestión ambiental con todas las actividades, productos y procesos de la empresa, reduciendo los impactos ambientales que puedan causar estos procesos y mejorar la actuación ambiental de la organización. Así mismo, los procesos ambientales parten del reto de detener y revertir el deterioro del medio ambiente con el fin de preservar y mejorar su calidad para las futuras generaciones, además de la necesidad de la sociedad de conservar y mejorar la oferta ambiental y la calidad ambiental de los recursos que satisfacen las necesidades de los seres humanos, y que son fundamentales para garantizar la vida [3]. Por ello, en esta investigación se propusieron algunos indicadores (ver tabla 1), mediante los que se evaluaron los procesos ambientales que se llevan a cabo en las empresas, y hacer el respectivo plan de mejoramiento ambiental por parte de las minas objeto de estudio dedicadas a la explotación de arcilla en La Guajira, estos resultados en la evaluación de los indicadores fueron los siguientes: En cuanto, a la viabilidad ambiental se encontró que los procesos mineros para la explotación de arcilla en La Guajira no cumplen con la legislación colombiana referente a títulos mineros y muy pocas se encuentra en el proceso de obtenerlos; en efecto, no se contempla un trabajo ambientalmente viable, ni la preocupación de las minas en ejecutar proyectos para tecnificar el proceso minero y/o trabajar de la mano con las comunidades, ni mucho menos realizar actividades post-mineria para mejorar las condiciones ambientales del área de influencia de explotación del mineral. Es decir, estas minas no presentan interés en incorporar estrategias para garantizar que los componentes bióticos, abiótico y socioeconómicos presentes en la zona se vean mínimamente afectados por la operación minera y de esta manera obtener títulos mineros que garanticen una mina sostenible. Así, en la búsqueda de evidencias en campo se encontró que en la mina “El Confuso”, Fonseca se intentó implementar un proyecto en el año 2015 para tecnificar la combustión del ladrillo, pero esta iniciativa no tuvo resultados significativos debido a que solo se quedó en la etapa de gestión por parte de las familias dueñas de las empresas mineras; este sería el único caso en las Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla minas objeto de estudio en donde se quiso evaluar la industrialización de uno de los procesos (quema de ladrillos). En este contexto, y para hacer que estas empresas sean viables ambientalmente se sugiere la implementación de proyectos que busquen la optimización de los procesos mineros y operaciones de las empresas, de esta manera se facilitaría el proceso de obtención de título minero y se motiva a los mineros de la región dedicados a la extracción de arcilla a implementar proyectos de industrialización en sus empresas mineras, con el fin de impulsar el uso integrado de los recursos naturales. En el caso de los conflictos ambientales, se encontró que actualmente la legislación minero-ambiental es excluyente y desarticulada ambientalmente hablando, el código de mina (ley 685 de 2001) no incluye la minería a menor escala, no crea condiciones para la obtención de títulos, no los acompaña en las transformaciones productivas y medio ambientales para que desarrollen la pequeña minería, también define una reglamentación propia para el manejo ambiental, desconociendo la Ley General Ambiental del país (Ley 99 de 1993) por lo tanto si la norma es flexible muchos simplemente no la cumplen, porque no se les está obligando. Además, según lo establecido en el artículo 332 de la Constitución Política de Colombia del 1991 donde: “El Estado es propietario del subsuelo y de los recursos naturales no renovables, sin perjuicio de los derechos adquiridos y perfeccionados con arreglo a las leyes preexistentes” y atendiendo a esto, se observa que desde la carta magna de la República de Colombia se genera el conflicto ambiental debido a que el subsuelo pertenece a la Nación; sin embargo, es explotado por dueños de los predios y/o tercero. En consecuencia, esta desarticulación está causando conflictos ambientales y territoriales entre la constitución Política, plan de ordenamiento territorial y código minero, causan un malestar al medio ambiente teniendo impactos significativos a largo plazo. Sin embargo, estas minas desde su informalidad pueden actuar de forma responsable, creando proyectos que ayuden en lo social y lo ambiental. Dentro de la valoración ambiental, se encontró que inicialmente la vocación de los suelos de las minas de arcilla objeto de estudio eran de uso forestal, donde se desarrollaban una serie de procesos que dieron lugar a ecosistemas ricos en biodiversidad, cosa que ha cambiado a medida que se ha extendido el área de operación de estas minas, impactando de forma negativa a los ecosistemas, generando una serie de problemáticas como la deforestación y disminución de la flora y la fauna. Además, se encontró que la minería dedicada a la explotación de arcilla en La Guajira se encuentra en auge, por lo que se proyecta la construcción de hornos y de excavaciones en nuevas áreas, por lo que el componente biótico de la zona será impactado directa e indirectamente en las diferentes etapas de los procesos mineros, dejando vulnerable al ecosistema a nuevas Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho interacciones y adaptaciones en las cuales aumentará la tasa de mortalidad. De esta manera se hace necesario implementar planes de manejo integrales en los que se enmarquen acciones enfocadas a garantizar un ambiente adecuado y sostenible para la comunidad y el medio ambiente. Finalmente, se encontró que la oferta ambiental genera preocupación, debido a que la minería de arcilla en La Guajira se practica ilegalmente, careciendo de un control efectivo por parte de las autoridades competentes para la regulación de la oferta ambiental disponible, por lo que ningún ente les impide realizar un uso inadecuado de estos, siendo las fases de descapote y extracción de arcilla las más influyentes en el desmejoramiento de la oferta ambiental, atentando contra la biodiversidad de especies presentes en la zona, la oferta del recurso hídrico y los bosques; por lo tanto, se vería afectada la capacidad de estos como sumideros y en síntesis, se perdería el contexto de desarrollo sostenible en las minas de arcilla de La Guajira. Pese a todo esto, se podría decir que los ecosistemas intervenidos por las empresas mineras, son capaz de recuperar su oferta ambiental, pero esto es un proceso tardío en el que a través del tiempo este se recupera progresivamente del daño causado, pero este proceso se puede potenciar si las empresas mineras se comprometen en realizar y aplicar un plan de postmineria que integre actividades que permitan compensar la afectación causada por la explotación de arcilla. Problemática ambiental Según [31], las problemáticas ambientales son cualquier alteración que ocasiona desequilibrio al medio ambiente, debido a actividades, procesos o comportamientos humanos, económicos, sociales, culturales y políticos, entre otros; que perturban el entorno natural y ocasionan impactos negativos sobre el medio ambiente, la economía y la sociedad, donde estas no pueden ser analizadas, ni entendidas, si no se tiene en cuenta una perspectiva global contextualizada, ya que surgen como consecuencia de múltiples factores que interactúan y se relacionan en función de la sociedad. En este sentido, las problemáticas ambientales son instancias en las que la forma de vivir de una sociedad determinada se ve alterada por los cambios en el entorno, los cuales afectan la salud humana a los que allí habitan y generan problemas al medio físico y biológico de los ecosistemas. Actualmente, en América Latina algunas de las problemáticas socioambientales más apremiante se relacionan a la cultura ambiental y el modelo extractivista, ya sean minería, agroindustria, pesca, fracking, entre otros. Estas problemáticas involucran muchos factores, entre ellos se incluyen diversos componentes, de los cuales se destacan dos aspectos, un componente de carácter social como lo son las migraciones, el aumento de pobreza, la pérdida de autonomía de comunidades locales y el otro aspecto sería el ambiental, tales como las emisiones de cargas contaminantes, la Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla extinción de especies, la degradación de ecosistemas y la pérdida de bosques y suelos [32,33]. Es así, como en esta investigación se estudió la problemática ambiental de las empresas mineras dedicadas a la explotación de arcilla en La Guajira, llevándose a cabo mediante una metodología para identificar las actividades que perturban el entorno natural y ocasionan impactos negativos sobre el medio ambiente, la economía y lo social. Además, de observar en campo indicadores como la cultura ambiental, cargas contaminantes, efectos a la salud y los problemas de medio físico, causadas por esta actividad no planificada. De esta manera, y en referencia a la Evaluación de Impacto Ambiental (EIA) se establecieron tres (3) momentos para evaluar las externalidades o impactos ambientales en las empresas mineras dedicadas a la explotación de arcilla en La Guajira, estos momentos se observan en la Figura 1. Figura 1. Momentos de evaluación ambiental en las minas que explotan arcilla en La Guajira A S PI FA RI ASP I 1. Caracterización del proyecto e Identificación de IA FAR I 2. EIA Rang os IA 3. Priorizacion del impacto Fuente: Elaboración propia a partir de [28] Donde, en el primer momento de evaluación se identificaron trece (13) ASPI: prospección, descapote, construcción de hornos, construcción de áreas operativas, extracción de arcilla, mezcla y amasado de arcilla, moldeado de ladrillos, secado de ladrillos, carga de ladrillo al horno, cocción de ladrillos, descarga del horno, despacho y traslado de ladrillos y por último, cierre y abandono de las minas, integradas en cinco (5) fases: exploración, construcción, explotación, operación y desmantelamiento y tres (3) FARI: sistema abiótico, biótico y antropogénico, identificando 19 impactos y teniendo en cuenta componentes ambientales como el recurso hídrico, suelo, aire, flora, fauna, medio social y económico de las empresas mineras objeto de estudio. A continuación, en la tabla 5 se muestra una síntesis de los resultados de la EIA en las empresas mineras dedicadas a la explotación de arcilla en La Guajira, obtenidos mediante la matriz de Leopold. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho Tabla 5. Matriz síntesis de los resultados de EIA en las empresas mineras que explotan arcilla en La Guajira Valor total FARI Impacto -3 Impacto Operación Agua Desmantelamiento Impacto Explotación Impacto Construcción Impacto Exploración 12 Sistema abiótico Agua -50 Agua -16 Agua Agua 42 0 Suelo 14 Suelo 5 Aire -28 14 Suelo 1 Aire -21 -147 27 Suelo -14 Suelo 56 0 Aire -64 -514 Aire -94 Aire 51 -156 Sistema biótico Flora 10 Flora 12 Flora Medio natural 4 Flora -60 Flora 8 16 1 -443 Fauna -12 Fauna -98 Fauna 12 Fauna -60 Fauna -71 0 -361 Sistema antrópico Económic Valor total ASPI Medio humano 10 - o -74 Económic o 37 Económi co 90 Económi 14 co 4 Social 10 ón 40 1 -90 Social -30 107 Social -39 Social -30 Social 2 Exploraci Económico 12 3 Construcci ón 40 2 Explotaci ón 62 2 Operació n 21 0 -324 Desmantelami ento - - 20 1.83 3 8 Fuente: Elaboración propia a partir de [ 28] Como se aprecia en la Tabla 5, el impacto ambiental generado por las minas de arcilla es de -1.838 unidades, el cual representa un 7.4% de externalidades ambientales negativas situando a esta actividad minera en un rango de impacto bajo, en comparación a los máximos resultados que se pueden obtener aplicando la matriz de Leopold, debido a que el número de FARI Y ASPI (19 FARI y 13 ASPI), y con un valor máximo de impacto de 100 unidades, se obtendría un valor total de impacto de -24.700 unidades (100%), como mayor grado posible de afectación a la calidad ambiental por parte de las empresas mineras dedicadas a la extracción de arcilla en La Guajira. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Se obtuvo, que con respecto a la puntuación que obtuvo el proyecto (-1.838 unidades), el medio con mayor afectación fue el natural con -1.621 unidades que corresponde así al 88.2% de afectación; en contraste, el medio humano presento una afectación negativa con - 217 unidades (11.8%) (Ver Figura 2). Por otro lado, el proceso minero de mayor afectación es la explotación del mineral arcilloso con -622 unidades, con un porcentaje de 33.8%. En cuanto a la afectación por componente ambiental, se encontró que el que el medio más afectado es el suelo con -514 unidades el cual representa el 28%, lo cual se debe a que el suelo sufre alteraciones en las fases de descapote, extracción y cierre u abandono de las minas, y, se obtuvo un impacto positivo en el componente económico (ver Figura 3). Figura 2. Impacto por medio que afecta las actividades en la minería de arcilla de La Guajira -1621 -217 MEDIO HUMANO MEDIO NATURAL 0 -200 -400 -600 -800 -1000 -1200 -1400 -1600 -1800 Fuente: Elaboración propia a partir de [28] Figura 3. Impacto por componente ambiental afectado en la minería de arcilla de La Guajira 200 FLORA FAUNA -400 -324 SOCIAL -361 ECONOM ICO -156 -443 AIRE SUELO -200 -147 AGUA 0 107 -514 -600 Fuente: Elaboración propia a partir de [28] En este sentido y de acuerdo con la metodología planteada, donde cada atributo de evaluación tiene un valor máximo de (10) y uno mínimo de uno (1) (ver Tabla 3), luego de aplicar la matriz de Leopold de EIA se obtuvo que los valores de impacto ambiental (IA) total de las ASPI oscilan entre -630 y 229 unidades y los FARI con valores que oscilaron entre -248 y 287 unidades, por ello con el fin de priorizar los IA que causan las actividades mineras (ASPI) y los factores ambientales receptores de impactos (FARI) se establecieron tres rangos que se presentan en la Tabla 6 y 7 respectivamente. Para ello se establece una clasificación de mayor a menor, en donde los valores obtenidos y, aquellos que sean menores, es decir con impacto negativo, resultarán como las actividades de mayor prioridad. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho Tabla 6. Rangos para priorización por ASPI de impacto ambiental en la minería de arcilla en La Guajira Rango IA Prioridad por ASPI Entre -630 y -343 Actividad de prioridad alta: se requieren medidas prioritarias urgentes para la prevención, mitigación, control, restauración y/o compensación del impacto ambiental generado y se asigna un color rojo. Entre -342 Actividad de prioridad media: se requieren medidas de manejo del impacto; sin embargo, y -57 estas no son prioritarias y se asigna un color naranja. Entre -56 y 229 Actividad de prioridad baja: las medidas no son prioritarias y se asigna un color verde. Fuente: Elaboración propia a partir de [ 28] Tabla 7. Rangos para priorización por FARI de impacto ambiental en la minería de arcilla en La Guajira Rango IA Entre Prioridad por FARI - 248 y -70 Impacto de prioridad alta: se requieren medidas prioritarias urgentes para la prevención, mitigación, control, restauración y/o compensación del impacto ambiental generado y se asigna un color rojo. Entre -69 Impacto de prioridad media: se requieren medidas de manejo del impacto; sin embargo, estas y 109 Entre 110 y 287 no son prioritarias y se asigna un color naranja. Impacto de prioridad baja: las medidas no son prioritarias y se asigna un color verde. Fuente: Elaboración propia a partir de [ 28] En este orden, se clasificaron las ASPI y los FARI de las empresas mineras dedicadas a la extracción de arcilla en La Guajira; generando un listado de acuerdo a una jerarquía basada en categorías, componentes ambientales y actividades mineras (Matriz de Leopold). Es importante anotar que, por razones de extensión en este artículo no se muestra la matriz de Leopold, pero está disponible contactando a los autores por correo electrónico. Los rangos para priorización de impacto ambiental, permitieron la jerarquización por impacto de las ASPI y los FARI (ver Tabla 8 y 9), permite que los impactos puedan ser intervenidos de acuerdo con el orden de prioridades, en donde: las de mayor impacto serán las que requieren intervención inmediata, seguidas de las de medio impacto y, por último, las de bajo impacto (ver Tabla 6 y 7). Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Tabla 8. Jerarquización por impacto de las ASPI en la minería de arcilla en La Guajira No. Impacto y jerarquización de las ASPI Impacto Impacto de prioridad alta 1 Descapote y/o remoción de capa vegetal -630 2 Extracción de arcilla -622 Impacto de prioridad media 3 Cocción de ladrillos -329 4 Construcción de hornos artesanales -277 5 cierre y abandono (Desmantelamiento/demolición) -210 6 construcción y adecuación de áreas operativas -125 Impacto de prioridad baja 7 Despacho y Traslado de los ladrillos 7 8 Secado de los ladrillos 11 9 Descargar ladrillos del horno 17 10 Carga de ladrillos al horno artesanal 23 11 Mezcla y amasado del mineral arcilloso 32 12 Moldeado de los ladrillos 36 13 Prospección y/o trabajos preliminares 229 Fuente: Elaboración propia a partir de [28] En la Tabla 8 se observa que de las 13 acciones de las empresas mineras susceptibles de producir impactos (ASPI), dos (2) de ellos son de alta prioridad ambiental, cuatro (4) son de significancia media y, siete (7) de significancia baja. Tabla 9. Jerarquización por impacto de los FARI en la minería de arcilla en La Guajira No. Impacto y jerarquización de los FARI Impacto Impacto de prioridad alta 1 Alteración y/o pérdida de corredores biológicos, hábitat y matriz de vegetación -248 2 Deterioro de la calidad del suelo -235 3 Afectación en la tenencia de la tierra por aumento de procesos erosivos, socavación y pérdida de suelo -180 4 Pérdida de abundancia y/o diversidad de especies animales terrestres, acuáticas -178 5 Pérdida de la calidad estética del paisaje -177 6 Daños al medio físico -145 7 Afectación a la calidad de vida de las comunidades -135 8 capacidad de uso y/o aumento en la ocurrencia de remoción del suelo -134 9 Pérdida de abundancia y/o diversidad de especies vegetales -124 Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho 10 Aumento/disminución de aves -112 11 Deterioro de la calidad del aire -94 12 Perdida de especies animales protegidas y/o en peligro de extinción -71 13 Perdida de especies vegetales protegidas y/o en peligro de extinción -71 Impacto de prioridad media 14 Aumento de los niveles de vibraciones o ruido ambiental -62 15 Deterioro de la calidad de agua subterránea -59 16 Deterioro de la calidad de agua superficial -56 17 Disminución del recurso hídrico -32 18 Incremento/disminución del índice demográfico -12 Impacto de prioridad baja 19 Generación de empleo 287 Fuente: Elaboración propia a partir de [ 28] Así mismo, en la Tabla 9 se observa que de los diecinueve (19) factores ambientales receptores de impactos (FARI), trece (13) de ellos son de alta prioridad ambiental, cinco (5) son de significancia media y, uno (1) de significancia baja en la minería de arcilla de La Guajira. Con respecto a los resultados obtenidos, se encontraron similitudes con lo expuesto por [22], cuyo trabajo fue un estudio sobre la evaluación de impacto ambiental en la mina artesanal de arcilla, Santa Cruz en el municipio de Manaure, La Guajira, por ello se deduce que los resultados son acertados y se acercan a la realidad ambiental por la que pasan las empresas mineras dedicadas a la extracción de arcilla en La Guajira. En este orden de ideas, cabe destacar que la información recolectada en las empresas de minería de arcilla de La Guajira (ver Tabla 2) fue de buena calidad, pero careció de datos de soporte para la evaluación de criterios como el número de habitantes con enfermedades causadas por las actividades mineras, la cantidad de agua consumida y la tasa de emisiones de gases de efecto invernadero y material particulado generados en sus procesos mineros; sin embargo, con la metodología utilizada en campo se muestran resultados significativos con los que se hizo un análisis profundo sobre indicadores como la cultura ambiental, emisión de cargas contaminantes, efectos a la salud humana, y problemas al medio físico (ver Tabla 1). De este modo, se detallan los resultados obtenidos del estudio de los indicadores, mediante la observación en campo sobre la problemática ambiental por las que pasa la minería de arcilla en La Guajira. Donde, se encontró que la cultura ambiental en la minería de arcilla en La Guajira no promueve la planeación, gestión y seguimiento ambiental que se Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla requiere; debido a que la relación del hombre con la naturaleza se da de forma irracional por falta de gobernanza y valores ambientales, ya que se observa que los trabajadores y/o familias dueñas de las empresas mineras no cuentan con el empoderamiento por el cuidado del medio ambiente. De esta manera, existe un patrón en el departamento de La Guajira entre las empresas objetos de estudios dedicadas a la extracción y comercialización de ladrillos (Confuso S.A, Loma Fresca S.A, Camarones S.A, La junta S.A, San Pedrito S.A), este patrón muestra que estas minas no cuentan con cultura ambiental. Hoy en día no se percibe un sentido ancestral que promueva una educación que genere el conocimiento para cuidar el medio ambiente y tener valores, hábitos, una conciencia sobre la problemática ambiental y/o un comportamiento para lograr un desarrollo sostenible. Por lo anterior, se hace necesario brindar conocimientos a la comunidad sobre conciencia ambiental, para cambiar los comportamientos y hábitos inadecuados que actualmente están generando el deterioro de los recursos naturales; de esta manera se podría garantizar un equilibrio Hombre– Naturaleza y crear generaciones futuras responsables con el cuidado del medio ambiente, promoviendo el desarrollo sostenible en estas minas. Para el caso de las cargas contaminantes en las minas de arcilla, se halló que se están realizando actividades de excavación en el terreno y quema de ladrillos en los hornos artesanales, que están generando cargas contaminantes, como material particulado (PM10 / PM2.5) y gases de efecto invernadero (GEI), afectando las fuentes hídricas y la atmosfera; donde, el impacto ambiental más significativo es la degradación de la capa de ozono, por efecto del humo de la quema de los ladrillos en los hornos, seguido de las cenizas que se producen en el horno y son arrojadas a las fuentes hídricas causando daños al cuerpo de agua. Además, se evidencio que todas las minas objeto de estudio aplican el mismo proceso para la producción de ladrillo, sin embargo, empresas mineras como “Loma Fresca S.A” y “San pedrito S.A” están implementando una estrategia en el proceso de excavación para reducir el material particulado, esta consiste en el riego previo del área que va a ser intervenida; pero ninguna de las minas visitadas contempla una técnica para disminuir los GEI producidos por la combustión de leña, utilizada en el proceso de deshidratación del ladrillo. Por otro lado, el manejo que se le está brindando a las cenizas no es el adecuado; sin embargo, se encontró que la mina “La Junta S.A”, utiliza las cenizas como abono para siembra de árboles y jardines. En este sentido, la recomendación es implementar tecnologías limpias que permitan disminuir la emisión de GEI generados en el proceso de combustión de la leña; de esta manera, se tecnifica el proceso de fabricación de ladrillos y se realiza un aporte significativo a favor del calentamiento global y por ende efectuando un acto de justicia a favor del cambio climático. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho Dentro de los efectos a la salud, se tuvo en cuenta que la minería de arcilla en La Guajira se aplica de forma rudimentaria, generando implicaciones a la salud humana de los trabajadores como, lesiones al cuerpo y enfermedades respiratoria, estas últimas transcienden a las comunidades aledañas, donde son los infantes quienes se ven principalmente afectados por el tiempo de exposición (1- 3 días) frente a las fumarolas generadas por los hornos artesanales. Pese a todo esto, en las minas no existe ni la mínima intención por parte de estas para aplicar medidas preventivas y/o correctivas para subsanar esta problemática socioambiental. También, se encontró que Las minas de arcilla objeto de estudio no se encuentran registradas ante el Ministerio de Minas y Energía, por lo que se trata de una actividad ilegal, en donde los trabajadores no portan ningún tipo de seguridad industrial y/o elementos de protección personal (EPP) que permitan combatir los riegos de accidente y perjuicios a la salud, cabe resaltar que las empresas no tienen el compromiso de dotar a los trabajadores de los EPP y es exclusivamente responsabilidad de los trabajadores, aun cuando los responsables de esta dotación son las empresas según lo establecido por el sistema de gestión de seguridad y salud en el trabajo de Colombia. Por ello se recomendó a las empresas mineras a darle solución a esta precaria situación, donde por lo menos los trabajadores contaran con las condiciones mínimas de seguridad y saneamiento, además de elementos para la protección personal y tecnologías limpias (blandas y duras) que permitan facilitar la explotación y manejo del mineral sin comprometer la integridad física de los trabajadores; así como subsanar las enfermedades respiratorias de la clase obrera y las comunidades aledañas. Por último, los problemas de medio físico generados por la explotación del mineral arcilloso, experimentan un crecimiento exponencial en función del uso indiscriminado de los suelos y por consiguiente el aumento de la deforestación, que más que un inconveniente en la fase de descapote, el problema subsiste en la manera de cómo se ejecuta el proceso de combustión del ladrillo, debido a que los árboles son cortados en grandes proporciones y aunado a la situación, después de terminada la explotación en un sitio especifico; es decir en la etapa de desmantelamiento y abandono, no se realiza una adecuación paisajística, dejando las excavaciones que al pasar del tiempo se rellenan por el arrastre de material en épocas de lluvias y se activa nuevamente la vegetación por acciones naturales. Así mismo, la flora se ha visto muy perjudicada debido a los efectos de la extracción y procesamiento de la arcilla, donde también se ve afectada en forma directa la fauna, ya que al ser removida la capa vegetal, los animales son desplazados de sus nichos ecológicos; un ejemplo de este comportamiento obedece a la mina de Camarones donde la intervención de las empresas mineras por la extracción del material ha generado un impacto significativo obligando a que los flamencos que anteriormente habitaban allí Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla migraran a otras zonas de la reserva natural; mientras que en la mina “La Junta S.A”. En este orden de ideas, se consideró importante recomendar a las empresas mineras que existan programas u actividades dentro de estas para reforestación de las áreas intervenidas; además de buscar acciones y/o estrategias para prevenir, mitigar, controlar y restaurar el suelo luego de ser explotado. Para tal efecto, debe existir un compromiso por todas las empresas dedicadas a la explotación y comercialización de arcilla, garantizando qué las zonas mineras no generen impactos a largo plazo al medio ambiente. Conclusiones Luego de realizado el análisis y discusiones de los resultados obtenidos sobre las tecnologías, procesos ambientales y problemática ambiental en la minería de arcilla en La Guajira colombiana; se considera, la importancia de concebir procesos mineros utilizando tecnologías limpias, los procesos ambientales tan complejos que generan diversas conjeturas en este tipo de minería, además de la creciente problemática ambiental que afecta la sostenibilidad de las empresas dedicadas a la minería de arcilla, se llega a las siguientes conclusiones por cada dimensión estudiada: Para el caso de las tecnologías utilizadas al llevar a cabo los procesos y/o actividades en la minería de arcilla en La Guajira, se encontró que la explotación del material arcilloso se realiza de forma rudimentaria con herramientas tecnológicas muy pobre; dicho de otra manera, no se cuenta con ningún tipo de maquinaria, ya que la minería de arcilla en La Guajira es totalmente artesanal, donde las herramientas utilizadas en sus procesos mineros consta de paladragas, azadones, palas, machetes, motosierras, baldes, amasado manual (con las manos), moldes de madera o de hierro, leña, carretillas y plásticos, cuyas tecnologías incrementan el esfuerzo físico de los trabajadores, repercuten en que exista una baja capacidad de producción en las empresas mineras y afectan los procesos ambientales y legales en las empresas mineras. En el caso de proponer procesos ambientales en la minería de arcilla de La Guajira, se concluye que la legislación minero-ambiental es excluyente y desarticulada, ya que el código de mina (ley 685 de 2001), no crea condiciones para la obtención de títulos, no los acompaña en las transformaciones productivas y medio ambientales para que desarrollen MiPymes y a su vez define una reglamentación propia para el plan de manejo ambiental, desconociendo la Ley General Ambiental de Colombia (Ley 99 de 1993). Esta desarticulación es la causa de conflictos ambientales y territoriales entre la constitución política, el plan de ordenamiento territorial y el código minero causan un malestar al medio ambiente, teniendo Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Danny Daniel López Juvinao, Luis Miguel Torres Ustate, Fabio Orlando Moya Camacho impactos significativos a corto, mediano y largo plazo. Sin embargo, estas minas desde su informalidad pueden actuar de forma responsable, implementado medidas u estrategias que ayuden en lo social y ambiental. En este contexto, se deduce que la viabilidad en la minería de arcilla de La Guajira, implica la incorporación de procesos relacionados con la sostenibilidad ambiental; razón por la cual, se sugiere el uso de tecnologías limpias aplicadas a las minas de arcilla, para la optimización de los procesos mineros y/u operaciones en estas, por consiguiente, se facilitara el proceso de obtención de título minero y se motiva a los mineros de la región dedicados a la extracción de arcilla para que implementen proyectos que utilicen tecnologías limpias en sus canteras, con el fin de impulsar el uso integrado de los recursos naturales y los procesos ambientales, cuyos procesos deben estar encaminados a implantar un plan de manejo integral en los que se enmarquen acciones para garantizar un ambiente seguro y sostenible para la comunidad y el medio ambiente. Por último, en el caso de identificar la problemática ambiental en la minería de arcilla de La Guajira, se logró caracterizar y clasificar las ASPI y los FARI de las empresas dedicadas a la minería de arcilla para el proceso de EIA; generar un listado de impactos ambientales conformado por trece (13) ASPI y diecinueve (19) FARI, de acuerdo una jerarquía del ambiente basada en categorías, componentes, componentes y medios; y, desarrollar un modelo de EIA conformados por cinco (5) empresas mineras que permite la mejora en las operaciones de estas y otras minas de este tipo, asentadas en La Guajira. Así mismo, se identificaron nueve (9) impactos ambientales en la minería de arcilla de La Guajira, las cuales afectan al medio físico y a la salud humana en las minas, de los cuales cinco (5) son impactos al medio físico, entre ellos: deterioro en la calidad del aire, ruidos y vibraciones, erosión del suelo, contaminación del agua e intervención a la fauna y flora y, cuatro (4) son impactos al medio socio económico que incluye la calidad de vida, cambio del valor paisajístico, seguridad social y generación de empleo, siendo estos dos últimos positivos, pero solo benefician a un sector muy limitado de las comunidades aledañas. De los impactos negativos al medio físico el más crítico es la pérdida del valor paisajístico, equivalente a la perdida de fauna y cobertura vegetal producto de la explotación irracional del suelo en las minas, seguido por la contaminación del aire generado por la constante quema del ladrillo afectando la calidad de vida y siendo una posible causa de los problemas de salud de los habitantes de las viviendas más cercanas a las ladrilleras y por último, se encuentran los recursos hídricos que son impactados en menor proporción. Revista Investigación e Innovación en Ingenierías, vol. 8, n°. 2, pp. 20 - 43, julio – diciembre 2020 DOI: https://doi.org/10.17081/invinno.8.2.3857 Tecnologías, procesos y problemática ambiental en la Minería de arcilla Referencias bibliográficas 1. IPCC, Climate Change 2014: Impacts, Adaptation, and Vulnerability. Part A: Global and Sectoral Aspects. 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https://openalex.org/W2417657804	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0156688&type=printable	English	null	Effects of Land Cover on the Movement of Frugivorous Birds in a Heterogeneous Landscape	PloS one	2,016	cc-by	10,036	Effects of Land Cover on the Movement of Frugivorous Birds in a Heterogeneous Landscape Natalia Stefanini Da Silveira1☯, Bernardo Brandão S. Niebuhr1☯, Renata de Lara Muylaert1☯, Milton Cezar Ribeiro1☯, Marco Aurélio Pizo2☯ Natalia Stefanini Da Silveira1☯, Bernardo Brandão S. Niebuhr1☯, Renata de Lara Muylaert1☯, Milton Cezar Ribeiro1☯, Marco Aurélio Pizo2☯ 1 Departament of Ecology, Spatial Ecology and Conservation Lab, Universidade Estadual Paulista Julio de Mesquita Filho, Rio Claro, São Paulo, Brazil, 2 Departament of Zoology, Universidade Estadual Paulista Julio de Mesquita Filho, Rio Claro, São Paulo, Brazil a1111 ☯These authors contributed equally to this work. * nat.stefanini@gmail.com ☯These authors contributed equally to this work. * nat.stefanini@gmail.com RESEARCH ARTICLE OPEN ACCESS Movement is a key spatiotemporal process that enables interactions between animals and other elements of nature. The understanding of animal trajectories and the mechanisms that influence them at the landscape level can yield insight into ecological processes and potential solutions to specific ecological problems. Based upon optimal foraging models and empirical evidence, we hypothesized that movement by thrushes is highly tortuous (low average movement speeds and homogeneous distribution of turning angles) inside forests, moderately tortuous in urban areas, which present intermediary levels of resources, and minimally tortuous (high movement speeds and turning angles next to 0 radians) in open matrix types (e.g., crops and pasture). We used data on the trajectories of two common thrush species (Turdus rufiventris and Turdus leucomelas) collected by radio telemetry in a fragmented region in Brazil. Using a maximum likelihood model selection approach we fit four probability distribution models to average speed data, considering short-tailed, long- tailed, and scale-free distributions (to represent different regimes of movement variation), and one distribution to relative angle data. Models included land cover type and distance from forest-matrix edges as explanatory variables. Speed was greater farther away from for- est edges and increased faster inside forest habitat compared to urban and open matrices. However, turning angle was not influenced by land cover. Thrushes presented a very tortu- ous trajectory, with many displacements followed by turns near 180 degrees. Thrush trajec- tories resembled habitat and edge dependent, tortuous random walks, with a well-defined movement scale inside each land cover type. Although thrushes are habitat generalists, they showed a greater preference for forest edges, and thus may be considered edge spe- cialists. Our results reinforce the importance of studying animal movement patterns in order to understand ecological processes such as seed dispersal in fragmented areas, where the percentage of remaining habitat is dwindling. Citation: Da Silveira NS, Niebuhr BBS, Muylaert RdL, Ribeiro MC, Pizo MA (2016) Effects of Land Cover on the Movement of Frugivorous Birds in a Heterogeneous Landscape. PLoS ONE 11(6): e0156688. doi:10.1371/journal.pone.0156688 Editor: Christopher A. Lepczyk, Auburn University, UNITED STATES Editor: Christopher A. Lepczyk, Auburn University, UNITED STATES Received: August 21, 2015 Accepted: May 18, 2016 Published: June 3, 2016 Copyright: © 2016 Da Silveira 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: © 2016 Da Silveira 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. Introduction Competing Interests: The authors have declared that no competing interests exist. Human-caused landscape modifications such as habitat loss, fragmentation, and land use deg- radation have negative impacts on biodiversity and ecosystem functions [1, 2, 3]. Besides hav- ing direct effects on individuals and ecological interactions, these modifications may also affect animal movement, a process that links individual behavior to biotic interactions. Thus, study- ing how animal movement may responds to such alterations can improve our understanding of population and community ecology in a changing world [4–5]. Movement features may mediate predation, mortality, and resource encounter rates in altered environments [6–7], comprising one of the key processes in the maintenance of animal popula- tions in fragmented landscapes [8]. The interaction among animal morphology, behavior and landscape structure produces different movement signatures [9–10], each of which distinctly influ- ence animals’ responses to human-caused modifications. For instance, forest-specialist species, which rely mainly on forest resources, tend to have their home range inside forested areas and avoid leaving forest fragments. On the other hand, habitat generalist species that use a wider range of resources are expected to be less restricted in space, using a variety of habitat types including those subject to intense human influence [6, 11, 12]. Comprehending how habitat generalist spe- cies move in fragmented areas is especially important to understand complex interactions, such as animal-mediated seed dispersal. For example, the movement of frugivorous birds may influence not only their own population connectivity but also plant population dynamics [13, 14, 15, 16, 17]. A landscape is typically composed of several landscape elements. One can usually classify the landscape using the patch-corridor-matrix model, which considers patches of habitat embedded in matrix and structurally connected by narrow tracts of habitat, the corridors. In scenarios of high fragmentation, the matrix is the most extensive and connected landscape ele- ment, playing an important role in the functioning of the entire landscape [18]. Theory predicts that habitat generalist species in heterogeneous landscapes with high-qual- ity, low-risk matrices have high movement probabilities (i.e. a high probability or per capita rate of leaving a current location or habitat patch [19, 20, 21, 22, 23]). Thus, these species may show weak responses to boundaries between two different types of vegetation or habitat patches. In low-risk matrices, habitat generalist species are not expected to avoid a specific veg- etation type when near boundaries, moving long distances in such landscapes [6]. Movement of Frugivorous Birds in a Heterogeneous Landscape Data Availability Statement: All relevant data are within the paper and available in the Supporting Information “S1 File”. Data Availability Statement: All relevant data are within the paper and available in the Supporting Information “S1 File”. Funding: This work was supported by Centro Nacional de Desenvolvimento Cientifico e tecnológico (http://http://www.cnpq.br/) (MAP and MCR); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (http://http://www.capes.gov.br/) (NSS); and Fundação de Amparo a pesquisa do Estado de São Paulo 2013/50421-2 (http://http://www. fapesp.br/) (MCR and MAP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 1 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Introduction Likewise, they tend to perform tortuous paths inside habitat patches, where food resources are abundant, and present straighter trajectories in open matrices such as grasslands or agricultural fields [6, 24, 25, 26, 27]. In the context of anthropogenic landscape changes generally characterized by habitat loss and reduced matrix quality, how animal movement characteristics change is a widely open question. What are the effects of land cover on the movement of common and supposed generalist animals? This second question is particularly important given the tendency of individuals to spend more time in the matrix, potentially facing increased mortality risk and reduced movement success [6]. Several common bird species that are habitat generalists in the extensively modified Atlantic Forest landscapes are suitable models to address such questions. The presence of habitat-generalist bird species in urban and suburban areas is common worldwide, probably due to their adaptation or low specificity for roosts and food resources (i.e. broad environmental tolerance) [28]. For example, 31% of Brazilian bird species have been recorded in urban areas [29]. Thus, urban and suburban landscapes can provide many resources for common and generalist birds, such as thrushes [30]. Moreover, thrushes may benefit more from suburban landscapes than from pastures, probably due to fewer resources in the latter compared to native forest fragments and urban environments [31]. Forest areas have an abundance of fruit trees and insects on which thrushes feed, and where they are relatively protected from most predators [32, 33, 34, 35]. In such resource-rich 2 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape environments, animals are expected to perform short displacements and present tortuous trajec- tories [6, 7, 24, 27]. In contrast, urban areas are constantly changing and suffer direct human influence. Additionally, resource availability may vary according to those changes, then in terms of availability of food resources in urban areas must differ from forest areas. [36, 37, 38]. Although urban areas provide food sources, due to the presence of lawns and fruit trees [39, 40, 41], nest predation and young mortality rates can be higher in urban environments and juveniles seek areas of native forest after leaving their nests [32, 33, 34]. Introduction Therefore, we hypothe- sized that movement by thrushes is highly tortuous (low average movement speeds and homo- geneous distribution of turning angles) inside forests, moderately tortuous in urban areas, which present intermediary levels of resources, and minimally tortuous (high movement speeds and turning angles next to 0 radians) in open matrix types (e.g., crops and pasture), based upon optimal foraging models [24–25] and empirical evidence [26–27] (Fig 1). Strong responses are represented by extremely high turning angles, suggesting the avoidance of or preference for a certain land cover type. Due to the habitat-generalist habit of thrushes, we did not expect strong responses in relation to forest edges [6]. Because the two thrush species inves- tigated (Tudus leucomelas and Turdus rufiventris) demonstrate some preference for edge areas we also hypothesized that thrushes would exhibit low mean speeds and long periods of time spent near the edges [30–42]. Expected responses of average speed and turning angles with land cover variables are explained and shown graphically in Fig 1. Ethic statement and survey permits We affirm that fieldwork did not involve endangered or protected species. In accordance with the environmental legislation of Brazil (norm N° 154/2007) we received authorization (N° 39790–1, authentication code N° 56882872) from the Brazilian Federal Environmental Agency (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis—IBAMA), for mist netting and tagging with radio transmitters from 06/2013 and 08/2014. For more details, please access the SISBIO (Sistema de Autorização e Informação em Biodiversidade), www. icmbio.gov.br/sisbio. In addition, the study was conducted on private land, in and around Sítio Moinho Velho and located adjacent properties. For further information, please contact one of the owners of Sítio Moinho Velho, Dr. Marco Aurélio Pizo. PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape Fig 1. Predictions for average speed and turning angles of thrushes' trajectories in a fragmented landscape. Alternative hypotheses are explained and shown graphically considering the effects of land cover variables, land cover type, and distance to forest edges. In hypothesis H1 for average speed, zero values represent the contact area between two land cover classes (i.e. the edge). doi:10.1371/journal.pone.0156688.g001 es in a fragmented landscape. Alternative hypotheses are d cover type, and distance to forest edges. In hypothesis H1 for ses (i.e. the edge). Study area The study area was located in rural areas of Itatiba, state of São Paulo, southeastern Brazil (22° 57'S, 46° 44'W). The region has a temperate tropical climate, with temperatures between 18°C and 25°C (annual average: 20.6°C) and a mountainous terrain formed by the Serra da Jurema ridge. Historically, the region was covered by semi-deciduous Atlantic rainforest [43], which was fragmented centuries ago to make room for pasture and agricultural production [31]. The Atlantic Forest is a highly fragmented tropical biome composed mainly of large tracts of pas- ture and agricultural crops as well as small forest fragments covering less than 16% of its origi- nal extent. It is also the most urbanized region in Brazil [44–45]. The study area is dominated by three distinct land cover types: a) forest fragments in differ- ent successional stages (fragment areas varying from 1 to 20 ha, some of which are completely isolated while others are connected by elongated forest corridors), mixed with old plantations of Eucalyptus, with a native understory (hereafter, forest), b) urban areas, consisting of residen- tial condominiums, farms, and ranches, and c) open matrix, that consist on pasture, crops, and are traversed by a few natural hedges (live fences; 4 to 12 m width, 150 to 400 m length) formed by lines of native trees, particularly species that produce small fleshy fruits (<5 mm in 3 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Fig 1. Predictions for average speed and turning angles of thrushes' trajectories in a fragmented landscape. Alternative hypotheses are explained and shown graphically considering the effects of land cover variables, land cover type, and distance to forest edges. In hypothesis H1 for average speed, zero values represent the contact area between two land cover classes (i.e. the edge). Fig 1. Predictions for average speed and turning angles of thrushes' trajectories in a fragmented landscape. Alternative hypotheses are explained and shown graphically considering the effects of land cover variables, land cover type, and distance to forest edges. In hypothesis H1 for average speed, zero values represent the contact area between two land cover classes (i.e. the edge). doi:10.1371/journal.pone.0156688.g001 4 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape diameter) dispersed by birds such as Caseariasy lvestris, Lithrae amollioides and Erythroxylum deciduum [46] (Fig 2). The natural hedges are primarily composed of native plants and were here also considered as forest habitat type (S1 Fig). Land cover composition of the entire study area included 51% pasture, 0.1% crops (mainly Citrus and corn fields), 14.9% urban areas, 1.1% water, 23.4% forest fragments, and 9.5% old plantations of Eucalyptus embedded in for- ested areas. We considered all non-forest cover types in the study area as potentially adequate habitat for the studied species due to their supposed tendency to use the space outside forests, especially during reproductive periods when fewer animals were observed in native forests [35]. To understand the influence of landscape on thrush movement, we classified the areas according to the most abundant land cover classes (forest, urban areas, and open matrices), but also in binary land cover classes (forest and non-forest). These two classifications allow us to explore whether thrushes recognize and respond to different landscape elements or if the typi- cal binary classification is sufficient to capture the variation in their movement. Radiotelemetry Fieldwork was done from June 2013 to June 2014. We focused on the species Turdus rufiventris and Turdus leucomelas (S1 Table), which were captured with mist nets positioned in the edge of selected fragments at three sites within the study area (Fig 2). A research permit granted by the Chico Mendes Institute for Conservation and Biodiversity (SISBIO research license N° 39790–1) supported the fieldwork activities. Radio transmitters (model A1080, ATS Advanced Telemetry Systems) were affixed to each captured individual as a backpack and weighed 1.9 g, less than 5% of the body weight of the animal [47] (see S1 Table for individual body weights). Altogether, we monitored ten individu- als: five T. leucomelas (two females and three males) and five T. rufiventris (two females and three males). We marked locations for individuals through triangulation of the bearings obtained by two teams of observers positioned at GPS-mapped stations (Model Garmin 60CSX, error up to 10 m in open field). We tried to collect location positions approximately every 20 min throughout a tracking session. We also observed locations recorded directly from visual contact with the tagged individuals. Given the occasional loss of radio signal due to the thrushes’ high mobility, and the use of visual contact data, bird trajectories had an irregular distribution of time among relocations. The landscape was mountainous, which made difficult the triangulation of the bearings among forest fragments. Moreover, transmitters were small, what can decrease the distance range of the signal. Therefore, many daily points were eventually lost because of diffi- culty to move around and capture the signal in this type of relief. We completed 180 days of sampling, which permitted us to gather 336 points of activity for the ten monitored individuals. The collected points of activity were found mostly near the edges of forested areas (S2 Fig). Data analysis The general protocol for animal movement studies is based on recording movement paths from direct observation or telemetry. The movement paths are analyzed as discretized trajecto- ries, which can be represented as a series of linear displacements between location fixes (step lengths) separated by angular shifts (turning angles) [48, 49, 50]. In some cases, movement rates or average speeds are reported instead of step lengths in fixed time intervals [49, 50, 51, 52]. Here we described thrush movement by their average speeds (in m/min) and relative turn- ing angles (in radians), the response variables of this study. To consider speeds in a biologically meaningful way, we excluded time ranges longer than two hours and intervals in which there 5 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape Fig 2. Study area encompasses the city limits of Itatiba, State of São Paulo, southeastern Brazil. The red points correspond to locations where T. rufiventris and T. leucomelas were captured. Land cover composition was: 51.0% pasture, 0.1% crops (mainly Citrus and corn fields), 14.9% urban areas, 1.1% water, 23.4% forest, 9.5% old plantations of Eucalyptus embedded in forested areas. Live fences are formed by lines of native trees, such as Casearia sylvestris, Lithraea mollioides and Erythroxyllum deciduum, located on different properties. Fig 2. Study area encompasses the city limits of Itatiba, State of São Paulo, southeastern Brazil. The red points correspond to locations where T. rufiventris and T. leucomelas were captured. Land cover composition was: 51.0% pasture, 0.1% crops (mainly Citrus and corn fields), 14.9% urban areas, 1.1% water, 23.4% forest, 9.5% old plantations of Eucalyptus embedded in forested areas. Live fences are formed by lines of native trees, such as Casearia sylvestris, Lithraea mollioides and Erythroxyllum deciduum, located on different properties. doi:10.1371/journal.pone.0156688.g002 was no data collection, such as during night. We corroborated this exclusion by applying the Pearson's correlation test to verify the relationship between time and speed within a range of two points. We observed small negative correlation between speeds and intervals (r = -0.18; t = -3.07; df = 273, p = 0.002); high speeds occurred only over short intervals. After excluding these locations, samples sizes were 275 for average speeds and 242 for turning angles (S1 Table). Robust methods such as state-space models [53–54] and step selection functions [55–56] have been developed to analyze movement patterns. Movement of Frugivorous Birds in a Heterogeneous Landscape of movement data, such as those recorded with GPS equipment or radio-tracking methods. However, small and highly mobile bird species such as Turdus thrushes are difficult to track continuously and accurately. Methods less dependent on abundant and continuous data have been used to assess land cover effects on movement [57–58]. These methods separate the anal- ysis into two parts: one to identify the scale and distribution of movement rates and turning angles, and another to estimate the effects of landscape structure on movement patterns. Here we used a maximum likelihood model selection approach to identify the probability distribu- tion (among short-tailed, long-tailed, and scale-free distributions) and the combination of covariates–land cover and distance to forest edges–that best explain variation in movement speed and turning angles of thrushes. Four probability distributions were used to fit average speeds: exponential, Weibull, Lévy, and truncated Lévy distributions. Exponential distributions are short-tailed and consider that animals perform trajectories within a characteristic spatial scale (individual displacement lengths are centered around a typical value) [59], so that their path resembles a Brownian motion with normal diffusion properties [25]. On the other hand, Lévy distributions are long- tailed power-law distributions with infinite variance, giving rise to super diffusive dynamics. They have scale-free properties, which means that they allow values for displacement lengths and speeds much greater than the mean values of the distribution. In practice, they can be rep- resented by clusters of short displacements with high tortuosity separated by some very long moves. Truncated Lévy (or bounded Pareto) distributions are modified versions which limit the maximum value of the distribution but still maintain a long-tail and super diffusion proper- ties over relatively large time extensions [25]. Weibull distributions are more flexible and can present short- or heavy-tails, or even a non-zero mode, depending on their parameters, which can represent differences in behavior or responses to environmental gradients. We believe that by considering these four families of probability distributions we could represent distinct com- binations of movement patterns to be fit to the data. To fit turning angles, we used a wrapped Cauchy distribution. Negative log-likelihood func- tions were built to fit each of these distributions to our data. After model fitting, we used Akaike Information Criterion corrected for small samples (AICc) and Akaike weights (w, relative likeli- hood of the model) to compare competing models [60]. PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Data analysis These methods require a continuous series 6 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 We considered that differences in AIC values less than two (ΔAICc 2) were equally plausible to explain the observed patterns, but that for models with similar AICc values, we gave priority to simpler models by parsimony. To identify how landscape variables affect movement, we assumed that one of the parameters of these distributions (λ for exponential, k for Weibull, and μ for wrapped Cauchy) was a function that varied depending on the combinations of variables (Fig 3). These models were also com- pared to no-effect models, where all parameters were constant, and to models considering the effects of sex and species, to make sure the variation in movement variables were indeed due to landscape covariates. For truncated Lévy distributions, only no-effect models were considered, since they inherently consider a greater variation in movement speed or step lengths. The landscape variables considered were: (i) distance to edge, the distance to the nearest for- est edge in absolute terms, considering edge as the transition zone between different land cover types; (ii) binary land cover, which refers to the land cover class of the location where each thrush displacement started, considering a binary classification of forest and non-forest; and (iii) land cover, which is equivalent to (ii) but refers to a three-level classification—forest areas, urban areas, and open matrices—separating the non-forest areas into two classes, urban areas and pasture/plantations. All models, comments on model fitting, and the scripts of model implementation are available in the supplementary material (S1, S2, S3, and S4 Texts). To check for the effects of individual variation and the small sample size for some individuals (S1 Table) on results, we also developed a hierarchical version of the most plausible model for 7 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape Fig 3. Set of distributions and their parameter settings used to explain variation in the response variables. Models used for the model selection approach to understand the contribution of landscape variables, sex, and species to thrush movement. Models M6-M9 considered additive effects of covariates, while models M10 and M11 consider their interaction. Y is the response variable. Movement of Frugivorous Birds in a Heterogeneous Landscape Fig 3. Set of distributions and their parameter settings used to explain variation in the response variables. Models used for the model selection approach to understand the contribution of landscape variables, sex, and species to thrush movement. Models M6-M9 considered additive effects of covariates, while models M10 and M11 consider their interaction. Y is the response variable. doi:10.1371/journal.pone.0156688.g003 average speeds, considering individuals as a random effect on intercepts. As the results were qualitatively similar, comments on individual variation, description of the model, and scripts are not shown here, but are available in supplementary material (S5 and S6 Texts). All analyses were performed using R 3.1.2 environment (R Dev. Core Team 2014) and bbmle [61] and ade- habitat assembly packages [62]. Movement of Frugivorous Birds in a Heterogeneous Landscape average speed for Turdus leucomelas and T. rufiventris. Plausible model in italic. K is the number of estimated s (relative likelihood of the model). Table 1. Competing models describing average speed for Turdus leucomelas and T. rufiventris. Plausible model in italic. K is the number of estimated parameters and the w is the Akaike weights (relative likelihood of the model). Response variable Distribution Models dAICc K w Average speed Exponential M11 Land cover classes * distance to edge 0.0 6 0.881 Exponential M10 Binary cover classes * distance to edge 4.1 4 0.114 Weibull M11 Land cover classes * distance to edge 13.1 7 0.001 Weibull M8 Species + binary cover classes 13.3 4 0.001 Weibull M9 Species +land cover classes 15.1 5 <0.001 Weibull M10 Binary cover classes * distance to edge 15.4 5 <0.001 Weibull M3 Species 17.3 3 <0.001 Exponential M9 Species + land cover classes 21.2 4 <0.001 Exponential M8 Species + binary cover classes 21.5 3 <0.001 Weibull M1 Binary cover classes 22.1 3 <0.001 Weibull M6 Sex + binary cover classes 22.5 4 <0.001 Exponential M5 Distance to edge 22.7 2 <0.001 Weibull M2 Land cover classes 24.2 4 <0.001 Weibull M5 Distance to edge 24.5 3 <0.001 Weibull M7 Sex + land cover classes 24.5 5 <0.001 Weibull M0 No effect model 26.0 2 <0.001 Weibull M4 Sex 26.2 3 <0.001 Exponential M3 Species 29.0 2 <0.001 Exponential M6 Sex + binary cover classes 34.3 3 <0.001 Exponential M7 Sex+ land cover classes 35.4 4 <0.001 Exponential M4 Sex 39.0 2 <0.001 Exponential M1 Binary cover classes 41.3 2 <0.001 Exponential M2 Land cover classes 42.6 3 <0.001 Exponential M0 No effect model 47.1 1 <0.001 Truncated lévy M0 No effect model 237.6 1 <0.001 Lévy M0 No effect model 319.4 1 <0.001 doi:10.1371/journal.pone.0156688.t001 Table 1. Competing models describing average speed for Turdus leucomelas and T. rufiventris. Plausible m parameters and the w is the Akaike weights (relative likelihood of the model). night period. The first displacement did not have a relative previous angle to be compared. The most plausible model in explaining the average movement speed was the exponential model (M11), which considered land cover (forest, open matrices, and urban areas) and forest edge distances influencing the pattern of average speeds and had an exponential error (Table 1). Results We recorded 275 displacements and 242 turning angles for the ten individuals of the two thrush species. The number of speed values and turning angles differ due to breaks in the tra- jectories, such as, when there was an interruption of the radio signal and at long intervals at the 8 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Regarding movement speed no other model was as plausible as (AICc difference < 2) or had comparable weight to M11. Average speeds increased as thrushes moved away from the forest edges either towards for- est interior or matrices (urban and other areas). Both the initial speeds and the rates of speed variation, increased for thrushes moving from the forest edges to the forest interior. Contrary to our expectations, the previous rates were higher than the rates for thrushes moving from the edges towards the matrices (Fig 4). Also, the average speeds were higher in the forest than in other habitats (open matrix and urban areas with approximate values; S3 Fig). Individuals pre- sented some variation in their response, but the qualitative response to landscape variables was similar when this variation was accounted for (S5 Text). In the case of turning angle, the model with the absence of effect was equally plausible to the others, but was the most parsimonious (Table 2). This show a weak or unpredictable effects of all selected predictors on turning angles, since both extrinsic (land cover, distance to forest edges) and intrinsic variables (sex, species) explain turning angle variation equally well. The 9 / 19 Movement of Frugivorous Birds in a Heterogeneous Landscape Fig 4. Average speeds of thrushes as a function of distances from edge, considering land cover classes. The dashed line represents the forest edges; green (forest), blue (open matrix—pastures and crops) and red (urban areas) lines represent the expected values for the mean speeds of individuals according to the exponential model M11, which considers the parameter settings being influenced by landscape variables (forest, open matrices and urban areas) and the forest edge distances. Speeds increase as individuals move away from forest edges. Fig 4. Average speeds of thrushes as a function of distances from edge, considering land cover classes. The dashed line represents the forest edges; green (forest), blue (open matrix—pastures and crops) and red (urban areas) lines represent the expected values for the mean speeds of individuals according to the exponential model M11, which considers the parameter settings being influenced by landscape variables (forest, open matrices and urban areas) and the forest edge distances. Speeds increase as individuals move away from forest edges. doi:10.1371/journal.pone.0156688.g004 doi:10.1371/journal.pone.0156688.g004 no-effect model shows that high turning angles are common, indicating a very tortuous move- ment pattern with many twists and relative angles close to 180 degrees, independent of land cover (Fig 5). In summary, trajectories of both thrush species, T. leucomelas and T. rufiventris, resembled habitat and edge dependent, tortuous random walks, with a well-defined movement scale inside each land cover type. Movement of Frugivorous Birds in a Heterogeneous Landscape ng angles for the trajectories for Turdus leucomelas and T. rufiventris. Plausible model in italic. K is the num- aike weights (relative likelihood of the model) and the w is the Akaike weights (relative likelihood of the model). Table 2. Competing models using turning angles for the trajectories for Turdus leucomelas and T. rufiventris. Plausible model in italic. K is the num- ber of estimated parameters and the w Akaike weights (relative likelihood of the model) and the w is the Akaike weights (relative likelihood of the model). Response Variable Models dAICc K w Turning Angles M0 No effect model 0.0 2 0.333 M1 Binary cover classes 1.9 3 0.130 M3 Species 1.9 3 0.129 M5 Distance to edge 2.0 3 0.122 M4 Sex 2.0 3 0.120 M8 Species + binary cover classes 3.8 4 0.049 M2 Land cover classes 3.9 4 0.047 M6 Sex + binary cover classes 3.9 4 0.046 M10 Binary cover classes * distance to edge 5.9 5 0.017 M9 Species +land cover classes 6.0 5 0.016 M7 Sex +land cover classes 6.2 5 0.014 M11 Land cover classes * distance to edge 10.0 7 0.002 doi:10.1371/journal.pone.0156688.t002 Table 2. Competing models using turning angles for the trajectories for Turdus leucomelas and T. rufiventris. Plausible model in italic. K is the num- ber of estimated parameters and the w Akaike weights (relative likelihood of the model) and the w is the Akaike weights (relative likelihood of the model). doi:10.1371/journal.pone.0156688.t002 plastic behavior and, considering thrushes’ ecological role, may translate into a great capacity for seed distribution over long distances among different landscape elements [23, 66, 67]. How- ever, the birds differed in average flight speed among landscape features: forest edges and land cover type (Table 1, Fig 4). Discussion Many variables can affect animals’ movement behavior and how it relates to interactions such as seed dispersal [63]. Intrinsic characteristics of the seed disperser (such as physiology and morphology), combined with extrinsic characteristics, particularly landscape structure and availability of food and shelter, influence the animals foraging mode and seed deposition over space and time [64–65]. In this study, thrush species were shown to perform very tortuous movement paths for all land cover types and landscape characteristics (Table 2, Fig 5). The thrushes’ movement speed changed with land cover, but the birds were able to move freely through the different habitat types in the landscape. These movement characteristics indicate a PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 10 / 19 Thrushes as habitat generalists or edge specialists? Compared to forest-specialist species, habitat generalist birds exhibit a greater propensity to move across or to use different habitat types without being significantly affected by changes in land cover. Forest-specialist species depend on large expanses of forest and high availability of resources [33]. However, being a habitat generalist does not imply a lack of habitat preferences. Instead, the habitat generalist is able to successfully modulate aspects of behavior in different habitat types. For example, juveniles of Turdus assimilis in Costa Rica had a higher survival rate in forested areas when compared to pastures and agricultural habitats, which may indicate a threshold amount of native forest providing critical resources for the persistence of the popu- lation [32]. Here we found low average speeds and a high frequency of observations near forest edges (Fig 4, S2 Fig), as well as a tendency to move faster far from such edges, although the thrushes used forest, open matrix and urban environments. This may represent an edge prefer- ence by T. leucomelas and T. rufiventris, together with the use different habitats. Therefore, they might be neither habitat generalists nor edge specialists only, but fall intermediate to both classifications. As in the present study, thrushes of the species T. rufiventris moved slowly near forest edges, but also spent considerable time in forest edges and were highly adapted to urban- ized regions in others studies [34–35]. Both cover types—forest edges and urban areas—may consist of foraging sites and, therefore, birds would pass quickly through forest interior regions to reach the edge areas. We also found very tortuous trajectories, with turning angles near 180°. This may represent “round trips” away and back to forest edges, reinforcing their tendency to stay next to them. Thrushes of the genus Turdus are similar in several aspects, including feeding habits and morphology [30–42]. This similarity translates to movement behavior, and may explain why our models that considered differences between species poorly explained observed trajectories, reinforcing the hypothesis that different species of the genus can be ecologically similar in terms of movement patterns. 11 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape Fig 5. Distribution of turning angles. Black points around the circle represent the relative angle observations. Note the high frequency of angle values of +180° or −180° (π or -π radians), which represents more abrupt turns and is characteristic of tortuous walks. PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Land cover types, resource distribution and movement Variability in thrush movement speed was best explained by an exponential distribution model and the expected value for speeds varied with land cover type. This pattern consisted of a char- acteristic scale of steps, lengths, and speeds for each land cover type, such that a thrush’s trajec- tory can be represented as a multi-scaled random walk in which the scale of speeds depends on habitat type [10, 25, 70]. Exponential signatures for animal movement have been observed in nomadic animals, which cover large areas in search of resources and do not settle in one single area [24–71]. The exponential signatures for movement was also observed in the present study for both species (maximum displacement = 1643.9 m/10 min, and minimum displace- ment = 2.82 m/15 min) and may relate to the availability of resources. This behavior, compris- ing steps with a well defined scale in places of high resource availability, has also been observed in other animals such as albatrosses (Diomedea exulans) [26], deer (Dama dama), and bees (Bombus terricola) [72]. As observed here, the scale of movement depends on habitat (and resource abundance), which is expected from theoretical models [70–73]. In fact, a theoretical models were tested with mud snails (Hydrobia ulvae), whose movement patterns responded to experimentally manipulated food availability [10]. However, the mud snail movements indi- cated that it is often hard to differentiate the evidence of a combination of exponential distribu- tions and of Lévy signatures. In the present study we did found evidence for exponential distributions instead of a Lévy distribution of average speeds, although this result may have been affected by methodological issues–thrushes (and therefore radio tags) are small, the radio signal is spatially limited, and the landscape terrain is rough, which turn difficult to record very long and fast displacements, characteristics of Lévy walks and flights [25]. We expected low average speeds in forests, followed by intermediate speeds in urban areas and higher speeds in pastures. Surprisingly, the results showed the opposite relationship, with a higher mean speed within the area containing the largest forest patch (223 ha) and a lower average speed in areas containing large amounts of other habitats. These other habitats were mainly formed by pastures and urbanized areas, which comprised 51% and 15% of the total study area, respectively. This unexpected pattern can be related to the foraging habits. Thrushes as habitat generalists or edge specialists? Fig 5. Distribution of turning angles. Black points around the circle represent the relative angle observations. Note the high frequency of angle values of +180° or180° (π or -π radians), which represents more abrupt turns and is characteristic of tortuous walks. doi:10.1371/journal.pone.0156688.g005 Using the same focal species, Vogel et al. [68] detected no interspecific differences in the use of habitat and forest strata in relation to the interior and edge of forest fragments, despite mor- phological differences, which corroborates our findings. However, when considering a broader assemblage, birds of the same genus (e.g. Turdus) may present different responses to landscape attributes in terms of movement [69]. When aspects of thrush behavior are combined to simu- late seed dispersal, they may present complementarity in the seed dispersal process [69], which has implications for the quantification of seed dispersal components. The difference between this study and ours may lie in the fact that the authors measured resource abundance itself, while we used land cover type as a proxy for resource and roost abundance. Our results support that the two studied species have similar movement patterns and consequently may exhibit similar roles as seed dispersers. Also, the tendency to stay next and move slower near forest edges may increase the probability of seed deposition by the studied thrush species in this micro habitat, what deserves further investigation. Still, the extent to which the similarity in movement showed here applies to other thrush species is still unknown, indicating a need to evaluate in other species. 12 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Conclusions Our study represented the first step in studying the fine scale movement of thrushes in Neo- tropical fragmented areas. Thrushes seem to be resilient and adapted to several forms of envi- ronmental degradation and showed a tendency to be edge specialists. Trajectories of thrushes resemble a highly tortuous habitat-dependent exponential distribution of movement speeds, representing a model of random walk with well-defined scales for each land cover type. These paths seem closely related to the foraging behavior of habitat-generalist animals, which have adapted to different environments, including urbanized areas. This result can deepen the understanding of animal responses to habitat fragmentation. Furthermore, the results can sup- port the management of remaining areas, as these frugivorous birds—potential dispersers of many plant species and among the main seed dispersers in Atlantic Forest [15–31]—move eas- ily over fragmentation gradients and should be considered in the development of management strategies for biodiversity conservation. We also suggest the integration of profiles for common frugivorous species into landscape resilience simulation models, particularly when planning the areas as set-asides for natural regeneration and predicting aspects of forest growth on a large scale [80, 81, 82], which is necessary for the restoration of the Atlantic Forest remnants. Movement of Frugivorous Birds in a Heterogeneous Landscape areas, live fences, and isolated trees in pasture or crop fields, due to the presence of fleshy fruits and invertebrates. Thus, the presence of these elements in fragmented areas favor plastic spe- cies such as Turdus but are less attractive to habitat-specialist species [75]. Also, this region has passed through an unusual drought, coinciding with the sampling period [76], which inhibited fruit production and may have led thrushes to find resources outside the forest, since fruit pro- duction was retarded (per. obs. by NSS). Predation is also a factor to be considered when analyzing animal movement. As common birds, thrushes of genus Turdus including T. rufiventris and T. leucomelas may commonly dis- play mobbing behavior, facing predators when other birds that are potentially a prey are aware of predation risk [77, 78, 79]. It was beyond our study to attempt tracking response to preda- tion risk, but overall we did not observe any predator attack on thrushes in any of the land cover types (per. obs. by NSS). The above discussion highlights the importance of noting the variety of search patterns emerging in habitats that contain a differential distribution of resources. Future research should include a spatially explicit model for resource distribution to measure organisms’ forag- ing trajectories and their possible outcomes, such as the number of seeds spread combined with information on foraging behavior. Investigating thrushes can improve our understanding of forest regeneration, and their role as seed dispersers may become even more perceptible in landscapes with low amounts of forest and in defaunated landscapes—where they are among the most common seed dispersers [15]. S1 Fig. Photo of the study area located in the countryside of Itatiba, São Paulo, Brazil. (PDF) S2 Fig. Frequency of activity points as a function of distance to forest edge. (PDF) Supporting Information S1 Fig. Photo of the study area located in the countryside of Itatiba, São Paulo, Brazil. (PDF) S1 Fig. Photo of the study area located in the countryside of Itatiba, São Paulo, Brazil. (PDF) Land cover types, resource distribution and movement Thrushes may also use common routes inside forest patches, especially when they have large home ranges, and tend to move quickly to forest edges, so that the speed inside forest is high. Thrushes have also been observed using living fences (per. obs. by NSS), narrow tracts of for- est-like environments that may function as corridors, inducing fast displacements over forest cover. Interestingly, due to the diversification of the environment, urban areas near forest frag- ments offer several features such as considerable roost and food resource availability that can benefit adaptable species [29]. A study [46] previously quantified the fruit availability in the hedges of the same study region, which appeared to be seasonal, peaking mostly in the period between September and February, and did not differ between forest patches and hedges. Similarly, no differences were observed between the amount of arthropods in fragments and hedges [46]. This information can also help us understand the slow speeds observed near the sampling point to the South, where pasture predominated and natural hedges connected forest fragments. Furthermore, our personal observations revealing that the secondary forests of the region are poor in fruits may also have contributed to the high speed of thrushes through forest interior, given the influence of vegetation type on the ease or success with which birds obtain resources [74]. Despite har- boring many living trees, several secondary forests seem to have equal or fewer food resources compared to orchards near urban settlements [46]. Secondary forests should, therefore, be less attractive to opportunistic frugivores, particularly in terms of foraging aspects and seasonal effects. Mainly during the dry season, thrushes may take advantage of orchards around urban 13 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Author Contributions Conceived and designed the experiments: NSS MCR MAP. Performed the experiments: NSS RLM. Analyzed the data: NSS BBSN RLM. Contributed reagents/materials/analysis tools: NSS BBSN MCR MAP. Wrote the paper: NSS BBSN RLM MCR MAP. Acknowledgments We are grateful to everyone who assisted us in data collection and technical support, to the owners of the areas where the studies were conducted, particularly at Sítio Moinho Velho, to M. Galetti for critically reading the manuscript, and to F. Martello for providing graphic sup- port on the images. We thank the anonymous reviewers for the invaluable suggestions for improving the manuscript. F. Barros, P. Rogeri, A. C. Silveira, F. S. Silveira and many others assisted us in the field. C. J. Leal and M. King for proofreading the manuscript. The Brazilian Research Council (CNPq), the Coordination for the Improvement of Higher Education Per- sonnel (CAPES) and São Paulo Research Foundation FAPESP (2013/50421-2) funded our study. MAP and MCR receive research grants from CNPq. S2 Fig. Frequency of activity points as a function of distance to forest edge. (PDF) S3 Fig. Influences of land use classes (a), sex (b) and species (c) on the average speeds of thrushes within fragmented landscapes of southeastern Brazil. (PDF) S3 Fig. Influences of land use classes (a), sex (b) and species (c) on the average speeds of thrushes within fragmented landscapes of southeastern Brazil. (PDF) 14 / 19 PLOS ONE \| DOI:10.1371/journal.pone.0156688 June 3, 2016 Movement of Frugivorous Birds in a Heterogeneous Landscape S1 File. Full data from two species of thrushes of the genus Turdus sp. collected by using the radio telemetry methodology from June 2013 to June 2014 in the city limits of Itatiba, São Paulo, Brazil. (PDF) S1 Table. Individuals of Turdus leucomelas and Turdus rufiventris followed by radio telem- etry from June 2013 to June 2014 in the city limits of Itatiba, São Paulo. (PDF) S1 Text. Movement analysis R script for thrushes in fragmented landscapes. (PDF) S2 Text. Description of the probability distributions used to explain variation in movement data. (PDF) S3 Text. Description of the models used to fit movement data. (PDF) S4 Text. Details of movement data fit. (PDF) S5 Text. Considerations of individual variation on the effects of land cover on thrush move- ment. (PDF) S6 Text. Hierarchical model script including individual variation. (PDF) 1. Fahrig L. Effects of habitat fragmentation on biodiversity. 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https://openalex.org/W2568981221	https://www.frontiersin.org/articles/10.3389/fpls.2016.02011/pdf	English	null	Editorial: Recent Advances in Flowering Time Control	Frontiers in plant science	2,017	cc-by	2,509	EDITORIAL published: 05 January 2017 doi: 10.3389/fpls.2016.02011 EDITORIAL published: 05 January 2017 doi: 10.3389/fpls.2016.02011 Recent Advances in Flowering Time Control The phenological development of plants can be broadly divided into 4 stages, embryo/juvenile, adult (all vegetative stages), reproductive (the generative stage), and senescent. This research topic focusses on the transition from vegetative growth to reproductive development, commonly referred to as ﬂoral transition. Plants have coordinated the seasonal timing of ﬂowering and reproduction with the prevailing environmental conditions. In agriculture, ﬂowering is a prerequisite for crop production whenever seeds or fruits are harvested. In contrast, avoidance of ﬂowering is necessary for harvesting vegetative parts of a plant such as tubers or roots. Late ﬂowering also severely hampers breeding success due to long generation times. Thus, ﬂowering time regulation is of utmost importance for genetic improvement of crops. In the past decades, we have gained increasing knowledge of ﬂowering time regulation in model species such as Arabidopsis thaliana (Blümel et al., 2015). Genes coordinately regulating ﬂoral transition have been grouped into diﬀerent pathways that have recently been illustrated in a WIKIPATHWAYS web interface (http://wikipathways.org//index.php?query=ﬂowering&title= Special%3ASearchPathways&doSearch=1&sa=Search). Several of these pathways are activated by diﬀerent environmental factors such as low temperature, day length, light intensity, or stress. Conservation of major ﬂowering-time regulators and regulatory pathways between diﬀerent species as well as increased availability of genome sequences and improvements in computational biology oﬀer a unique opportunity to study ﬂowering time genes across species. In general terms, the central elements that perceive day-length signals to control ﬂoral transition are conserved across the plant kingdom. CONSTANS (CO)-like genes and PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN (PEBP) encoding genes play major roles in these pathways and were ﬁrst identiﬁed by genetic analysis in Arabidopsis. CO-like sequences seem to exist in all plants. Recent evidence indicates that CO of Arabidopsis arose from a family-speciﬁc duplication and similar events might have occurred independently in many plant families (Simon et al., 2015). In contrast, plants from the Amaranthaceae family are lacking a true CO ortholog (Dally et al., 2014). Christian Jung 1, Klaus Pillen 2, Dorothee Staiger 3, George Coupland 4 and Maria von Korff 4, 5 Christian Jung 1, Klaus Pillen 2, Dorothee Staiger 3, George Coupland 4 and Maria von Korff 4, 5 1 Plant Breeding Institute, Christian-Albrechts-University of Kiel, Kiel, Germany, 2 Plant Breeding Institute, Martin Luther University of Halle-Wittenberg, Halle, Germany, 3 Molecular Cell Physiology, Faculty of Biology, Bielefeld University, Bielefeld, Germany, 4 Department of Plant Developmental Biology, Max Planck Institute for Plant Breeding Research, Cologne, Germany, 5 Cluster of Excellence in Plant Sciences, Heinrich-Heine-University Düsseldorf, Düsseldorf, Germany Keywords: ﬂoral transition, crop plants, Arabidopsis, phenological development, yield, evolution Edited and reviewed by: Neelima Roy Sinha, University of California, Davis, USA Correspondence: Christian Jung c.jung@plantbreeding.uni-kiel.de Edited and reviewed by: Neelima Roy Sinha, University of California, Davis, USA Correspondence: Christian Jung c.jung@plantbreeding.uni-kiel.de Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Specialty section: This article was submitted to Plant Evolution and Development, a section of the journal Frontiers in Plant Science Received: 15 October 2016 Accepted: 19 December 2016 Published: 05 January 2017 This research topic is focused on ﬂowering time control in cultivated species. It contains nine review, perspective, and opinion articles and 14 original research articles which cover a large range of organisms from model species to crops. Citation: Jung C, Pillen K, Staiger D, Coupland G and von Korff M (2017) Editorial: Recent Advances in Flowering Time Control. Front. Plant Sci. 7:2011. doi: 10.3389/fpls.2016.02011 New components have been added to the network of ﬂowering time regulators mostly working upstream of key regulator elements, e.g., GATA transcription factors, small RNAs, in particular microRNAs (miRNAs) or sugar molecules. The GATA transcription factors GATA, NITRATE-INDUCIBLE, CARBON-METABOLISM INVOLVED (GCN), and GCN-like (GNL) previously identiﬁed as growth regulators mediating control by several phytohormones have January 2017 \| Volume 7 \| Article 2011 Frontiers in Plant Science \| www.frontiersin.org 1 Editorial: Recent Advances in Flowering Time Control Jung et al. emerged as repressors of ﬂowering, acting via SOC1 (Richter et al., 2013; Behringer and Schwechheimer). During the juvenile to adult phase transition, a gradual decrease in miR156 and a reciprocal increase in miR172 ultimately leads to the activation of FLOWERING LOCUS T (FT) (Wang et al., 2009a; Wu et al., 2009). To facilitate the genome-wide analysis of small RNA-seq data, the DARIO tool developed for animals has been adapted for use in plants (plantDARIO) (Patra et al.). in this research topic (Fjellheim et al.). The authors discuss molecular pathways that control seasonal ﬂowering responses in the Pooideae sub-family and how variations in ﬂowering time gene activities contributed to the adaptation to diﬀerent environments. Reﬁned ﬂowering time regulatory pathways have been identiﬁed from rice (Shrestha et al., 2014) and barley/wheat (Chen et al., 2014) (Mulki and von Korﬀ, 2016). Loscos et al. show that natural allelic variation in copy number of the ﬂorigen HvFT1 is present in European spring barley cultivars lacking a vernalization requirement to initiate ﬂowering (Loscos et al., 2014). However, no clear relationship between HvFT1 copy number and expression was observed in a set of diverse spring barley genotypes. p (p ) ( ) The research topic also reﬂects the immense technical progress from the past years. Initially, ﬂowering time regulators from crops were mainly cloned due to their sequence homology with known ﬂowering time genes, mainly from Arabidopsis although the INDETERMINATE ﬂowering gene was cloned from maize by transposon tagging (Colasanti et al., 1998). Later, new genes were identiﬁed from crops using ﬂowering time QTLs (quantitative trait loci) by map-based cloning approaches. Now, whole genome or candidate gene association mapping and transcriptome analysis have become important strategies (Schiessl et al.). Citation: For instance, whole transcriptome analysis revealed the circadian clock homolog of EARLY FLOWERING 3 (ELF3) and mapping-by-sequencing applied on exome-capture data from phenotypic bulks identiﬁed PHYTOCHROME C as important components of photoperiodic ﬂowering in barley (Faure et al., 2012; Pankin et al., 2014). In addition, the wild barley nested association mapping population HEB-25 was used to associate major ﬂowering time genes with phenological development in diﬀerent ﬁeld environments (Maurer et al., 2015, 2016). Furthermore, several ﬂowering time genes like Ppd- H1 (PRR37) and HvSDW1 (GA20ox2) were linked to both, developmental and yield-related traits. In a salinity tolerance study with HEB-25, the wild barley allele at the HvCEN locus (Antirrhinum CENTRORADIALIS, TFL1-like) promoted ﬂowering and maturity, resulting in a higher harvest index and a higher yield under salt stress in the ﬁeld (Saade et al., 2016). These ﬁndings indicate that searching for allelic variants of known ﬂowering time genes, also taken from related wild species, may substantially support future plant breeding eﬀorts to increase plant performance under optimal cultivation conditions as well as under stress. Some articles from this research topic highlight multiple functions of ﬂowering time genes beyond ﬂoral transition. These genes impact multiple developmental processes and they are regulators of yield components and stress tolerance (Kazan and Lyons, 2016). In this respect, members of the PHOSPHATIDYLETHANOLAMINE-BINDING PROTEIN (PEBP) gene family, such as FLOWERING LOCUS T (FT) and TERMINAL FLOWER 1 (TFL) of Arabidopsis attracted the highest attention. As highlighted for tomato (Lifschitz et al.) and for rice (Izawa et al., 2016) ﬁne tuning of the SFT/SP (which are true orthologs of FT and TFL) ratio is an important process for patterning plant architecture. Lifschitz et al. explain how the ratios between FT-like and TFL1- like genes control the patterning of the shoot systems across many diﬀerent plants. In tomato, an increase of the ﬂorigen protein SINGLE FLOWER TRUSS (SFT, FT-like), relative to the anti-ﬂorigen protein SELF PRUNING (SP, TFL1-like) induces growth arrest and termination of meristems across the tomato shoot, while high relative levels of SP promote the formation of an indeterminate vegetative inﬂorescence. Consequently, SFT/SP ratios determine the number of ﬂowers and eventually, tomato fruits per inﬂorescence. Naturally occurring mutations have been selected in both gene families to adapt crops to diﬀerent environments and to increase productivity. TFL1 mutants have been frequently used in breeding (e.g., tomato, soybean, roses, and barley). Frontiers in Plant Science \| www.frontiersin.org Citation: Likewise, mutations of FT-like genes were selected in sunﬂower, sugar beet, rice, potato, and wheat. Moreover, single point mutations within FT- and TFL1 orthologs can drastically alter their function from ﬂoral inducers to ﬂoral repressors, as demonstrated for sugarcane Coelho et al. and beet (Pin et al., 2010). We propose that selecting for FT/TFL1 sequence variations in crop plants may pave the way to further improvements in elite crop productivity. Flowering time regulation is strongly conserved among the Brassicas to which Arabidopsis also belongs. Two articles (Guo et al.; Schiessl et al.) describe ﬂowering time genes from oilseed rape where the vernalization pathway with its central element FLOWERING LOCUS C (FLC) is essentially the same as in Arabidopsis. In contrast, an FLC homolog from beet was proven not to be a major regulator of vernalization response in biennial beets Vogt et al. New sequence variation has been induced in rapeseed by EMS mutagenesis which gave rise to plants with altered ﬂowering time in spite of the polyploid nature of this species. Mutations within a single gene can have a big impact on ﬂowering time even if there are several paralogs of an Arabidopsis ﬂowering time gene present in the rapeseed genome. AUTHOR CONTRIBUTIONS All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication. The research topic demonstrates that the range of model species has been constantly increased to allow a broader range of ﬂowering-related traits to be studied. Arabis alpina and Brachypodium distachyon serve as models for perennials (Wang et al., 2009b) and for grasses Woods et al., respectively. A recent overview on ﬂowering regulation in grass species is given REFERENCES Pin, P. A., Benlloch, R., Bonnet, D., Wremerth-Weich, E. S. I., Kraft, T., Gielen, J. J., et al. (2010). An antagonistic pair of FT homologs mediates the control of ﬂowering time in sugar beet. Science 330, 1397–1400. doi: 10.1126/science.1197004 Blümel, M., Dally, N., and Jung, C. (2015). Flowering time regulation in crops — what did we learn from Arabidopsis? Curr. Opin. Biotechnol. 32, 121–129. doi: 10.1016/j.copbio.2014.11.023 Richter, R., Behringer, C., Zourelidou, M., and Schwechheimer, C. (2013). Convergence of auxin and gibberellin signaling on the regulation of the GATA transcription factors GNC and GNL in Arabidopsis thaliana. Proc. Natl. Acad. Sci. U.S.A. 110, 13192–13197. doi: 10.1073/pnas.13042 50110 Chen, A., Li, C., Hu, W., Lau, M. Y., Lin, H., Rockwell, N. C., et al. (2014). PHYTOCHROME C plays a major role in the acceleration of wheat ﬂowering under long-day photoperiod. Proc. Natl. Acad. Sci. U.S.A. 111, 10037–10044. doi: 10.1073/pnas.1409795111 Saade, S., Maurer, A., Shahid, M., Oakey, H., Schmöckel, S. M., Negrão, S., et al. (2016). Yield-related salinity tolerance traits identiﬁed in a nested association mapping (NAM) population of wild barley. Sci. Rep. 6: 32586. doi: 10.1038/srep 32586 Colasanti, J., Yuan, Z., and Sundaresan, V. (1998). The indeterminate gene encodes a zinc ﬁnger protein and regulates a leaf-generated signal required for the transition to ﬂowering in maize. Cell 93, 593–603. doi: 10.1016/s0092-8674(00)81188-5 Shrestha, R., Gómez-Ariza, J., Brambilla, V., and Fornara, F. (2014). Molecular control of seasonal ﬂowering in rice, arabidopsis and temperate cereals. Ann. Bot. 114, 1445–1458. doi: 10.1093/aob/mcu032 Dally, N., Xiao, K., Holtgräwe, D., and Jung, C. (2014). The B2 ﬂowering time locus of beet encodes a zinc ﬁnger transcription factor. Proc. Natl. Acad. Sci. U.S.A. 111, 10365–10370. doi: 10.1073/pnas.1404829111 Simon, S., Rühl, M., De Montaigu, A., Wötzel, S., and Coupland, G. (2015). Evolution of CONSTANS regulation and function after gene duplication produced a photoperiodic ﬂowering switch in the brassicaceae. Mol. Biol. Evol. 32, 2284–2301. doi: 10.1093/molbev/msv110 Faure, S., Turner, A. S., Gruszka, D., Christodoulou, V., Davis, S. J., von Korﬀ, M., et al. (2012). Mutation at the circadian clock gene EARLY MATURITY 8 adapts domesticated barley (Hordeum vulgare) to short growing seasons. Proc. Natl. Acad. Sci. U.S.A. 109, 8328–8333. doi: 10.1073/pnas.1120496109 Acad. Sci. U.S.A. 109, 8328–8333. doi: 10.1073/pnas.112049610 Izawa, T., Okada, R., Endo, N., Nemoto, Y., Takamizo, T., Tsuzuki, S., et al. (2016). Poaceae Plant whose Flowering Time is Controllable. Tsukuba: United States Patent. Wang, J. REFERENCES W., Czech, B., and Weigel, D. (2009a). miR156-regulated SPL transcription factors deﬁne an endogenous ﬂowering pathway in Arabidopsis thaliana. Cell 138, 738–749. doi: 10.1016/j.cell.2009. 06.014 Kazan, K., and Lyons, R. (2016). The link between ﬂowering time and stress tolerance. J. Exp. Bot. 67, 47–60. doi: 10.1093/jxb/erv441 Wang, R. H., Farrona, S., Vincent, C., Joecker, A., Schoof, H., Turck, F., et al. (2009b). PEP1 regulates perennial ﬂowering in Arabis alpina. Nature 459, U423–U138. doi: 10.1038/nature07988 Loscos, J., Igartua, E., Contreras-Moreira, B., Gracia, M. P., and Casas, A. M. (2014). HvFT1 polymorphism and eﬀect-survey of barley germplasm and expression analysis. Front. Plant Sci. 5:251. doi: 10.3389/fpls.2014.00251 Wu, G., Park, M. Y., Conway, S. R., Wang, J. W., Weigel, D., and Poethig, R. S. (2009). The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138, 750–759. doi: 10.1016/j.cell.2009. 06.031 Wu, G., Park, M. Y., Conway, S. R., Wang, J. W., Weigel, D., and Poethig, R. S. (2009). The sequential action of miR156 and miR172 regulates developmental Maurer, A., Draba, V., Jiang, Y., Schnaithmann, F., Sharma, R., Schumann, E., et al. (2015). Modelling the genetic architecture of ﬂowering time control in barley through nested association mapping. BMC Genomics 16:290. doi: 10.1186/s12864-015-1459-7 timing in Arabidopsis. Cell 138, 750–759. doi: 10.1016/j.cell.2009. 06.031 Maurer, A., Draba, V., and Pillen, K. (2016). Genomic dissection of plant development and its impact on thousand grain weight in barley through nested association mapping. J. Exp. Bot. 67, 2507–2518. doi: 10.1093/jxb/erw070 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. sociation mapping. J. Exp. Bot. 67, 2507–2518. doi: 10.1093/jxb/er Mulki, M. A., and von Korﬀ, M. (2016). CONSTANS controls ﬂoral repression by up-regulating VERNALIZATION2 (VRN-H2) in barley. Plant Physiol. 170, 325–337. doi: 10.1104/pp.15.01350 Copyright © 2017 Jung, Pillen, Staiger, Coupland and von Korﬀ. 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. Pankin, A., Campoli, C., Dong, X., Kilian, B., Sharma, R., Himmelbach, A., et al. (2014). FUNDING We gratefully acknowledge funding by the German Research Foundation (Priority Program 1530 and Deutsche Forschungsgemeinschaft). January 2017 \| Volume 7 \| Article 2011 2 Editorial: Recent Advances in Flowering Time Control Jung et al. January 2017 \| Volume 7 \| Article 2011 REFERENCES Mapping-by-sequencing identiﬁes HvPHYTOCHROME C as a candidate gene for the early maturity 5 locus modulating the circadian clock and photoperiodic ﬂowering in barley. Genetics 198, 383–396. doi: 10.1534/genetics.114.165613 January 2017 \| Volume 7 \| Article 2011 Frontiers in Plant Science \| www.frontiersin.org 3
https://openalex.org/W4283766173	https://sea.ufr.edu.br/SEA/article/download/1553/1625	English	null	Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage	Scientific Electronic Archives	2,022	cc-by	9,585	Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Scientific Electronic Archives Issue ID: Sci. Elec. Arch. Vol. 15 (7) July 2022 DOI: http://dx.doi.org/10.36560/15720221553 Article link: https://sea.ufr.edu.br/SEA/article/view/1553 Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Corresponding author Gil Ignácio Cañizares Instituto Federal do Rio Grande do Sul, Bento Gonçalves Helen Fernanda Barros Gomes Universidade Federal de Rondonópolis gomes.helen@ufr.edu.br Raquel Vasconcelos Lourençon Lincoln University of Missouri - EUA Raquel Ornelas Marques Universidade Estadual Paulista - Campus de Botucatu Evelyn Prestes Brito Universidade Estadual Paulista - Campus de Botucatu Oscar Boaventura Neto Universidade Federal de Alagoas - Campus de Maceió Marleide Cañizares Instituto Federal do Rio Grande do Sul, Bento Gonçalves Heraldo Cesar Gonçalves Universidade Estadual Paulista - Campus de Botucatu ______________________________________________________________________________________ Abstract. An experiment was carried out to evaluate the replacement of corn silage with fresh sugarcane, at a roughage:concentrate ratio of 40:60, on the intake, dry matter (DM) and nutrient digestibility, milk yield and composition, and feeding behavior of lactating goats. Eight goats were distributed into two balanced 4 × 4 Latin squares, where the replacement levels of 0, 33, 67, and 100% were used as independent variables. Dry matter intake, which averaged 2.302 kg/day, showed a quadratic response with the minimum value observed at the sugarcane inclusion level of 68.04%. The treatments did not affect the intakes of crude protein (CP), neutral detergent fiber (NDF), or net energy, whereas non- Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Scientific Electronic Archives Issue ID: Sci. Elec. Arch. Vol. 15 (7) July 2022 DOI: http://dx.doi.org/10.36560/15720221553 Article link: https://sea.ufr.edu.br/SEA/article/view/1553 Scientific Electronic Archives Issue ID: Sci. Elec. Arch. Vol. 15 (7) July 2022 DOI: http://dx.doi.org/10.36560/15720221553 Article link: https://sea.ufr.edu.br/SEA/article/view/1553 Corresponding author Gil Ignácio Cañizares Instituto Federal do Rio Grande do Sul, Bento Gonçalves Corresponding author Gil Ignácio Cañizares p g Gil Ignácio Cañizares Instituto Federal do Rio Grande do Sul, Bento Gonçalves Oscar Boaventura Neto Universidade Federal de Alagoas - Campus de Maceió Oscar Boaventura Neto Universidade Federal de Alagoas - Campus de Maceió Marleide Cañizares Instituto Federal do Rio Grande do Sul, Bento Gonçalves ______________________________________________________________________________________ Abstract. An experiment was carried out to evaluate the replacement of corn silage with fresh sugarcane, at a roughage:concentrate ratio of 40:60, on the intake, dry matter (DM) and nutrient digestibility, milk yield and composition, and feeding behavior of lactating goats. Eight goats were distributed into two balanced 4 × 4 Latin squares, where the replacement levels of 0, 33, 67, and 100% were used as independent variables. Dry matter intake, which averaged 2.302 kg/day, showed a quadratic response with the minimum value observed at the sugarcane inclusion level of 68.04%. The treatments did not affect the intakes of crude protein (CP), neutral detergent fiber (NDF), or net energy, whereas non- fibrous carbohydrates (NFC) and total digestible nutrients (TDN) intakes responded quadratically and ether extract (EE) intake decreased with the increasing sugarcane levels. The levels of replacement of corn silage with sugarcane did not influence the digestibility coefficients of DM, CP, NDF, or NFC, but EE digestibility exhibited had a quadratic response. There were no significant differences for milk yield, which averaged 1.512 kg/day, even when yield was corrected for 3.5% fat; or the concentrations of protein, fat, lactose, total solids, and solids-not-fat in milk. Milk urea nitrogen, on the other hand, increased with the sugarcane levels. Fresh sugarcane can replace corn silage in the diet of low-yielding goats with a 40:60 roughage:concentrate ratio, as it will not change milk yield. Key words: crude protein, dry matter, milk production, rumen,urea Introduction In dairy goat farming, the low availability of feed—roughage, mainly—during times of scarcity concerns producers to stock feed, either preserved or in the form of grass for cutting, to ensure year- round productivity and stability in milk production. In this respect, fresh sugarcane constitutes a feed alternative for these periods, as it helps to maintain the milk production of goats while contributing for feed costs not to increase. The main advantage of sugarcane over other forage species is that it reaches its greatest forage potential and high levels of soluble sugars in the dry season. However, as a disadvantage, when used as exclusive feed or at high percentages in ruminant diets, its low protein and minerals levels and high fiber content impair digestibility. The experiment was laid out in a Latin square design. The animals were distributed into two 4 × 4 Latin squares according to milk yield (MY), to evaluate the levels of replacement (dry matter [DM] basis) of corn silage with chopped fresh sugarcane, at the rates of 0, 33, 67, and 100%. Treatments were thus as follows: 0% sugarcane + 100% corn silage; 33% sugarcane + 67% corn silage; 67% sugarcane + 33% corn silage; and 100% sugarcane + 0% corn silage (Table 1). The experimental diets were previously formulated, according to the NRC (2007), to meet the nutritional requirements of lactating goats with a MY potential of 2.5 kg/day, with a protein content of 15% (DM basis) (Table 2). Sugarcane may cause a reduction in intake due to its low fiber digestibility, as its average fiber content, of 54.00%, is lower than the 60.00% of silage (Campos et al., 2010). The digestibility of this feedstuff does not determine voluntary intake per se, but the physical nature of the diet and its ability to efficiently stimulate rumen function by supplying bypass nutrients, especially protein, are also contributing factors (Preston, 1982). If included in a diet, sugarcane must be accompanied by a concentrate that allows an adequate nutrient intake to meet the energy needs of production animals. Seedlings of sugarcane variety RB 72454 were acquired from sugarcane fields in the region, for planting. The corn belonged to variety Ag 4051, classified as a dent-texture hybrid, which was ensiled in a bag silo. The sugarcane was chopped daily and incorporated into the corn silage and concentrate forming a complete mixture, observing a roughage:concentrate ratio of 40:60 (DM basis). Heraldo Cesar Gonçalves Universidade Estadual Paulista - Campus de Botucatu ______________________________________________________________________________________ Abstract. An experiment was carried out to evaluate the replacement of corn silage with fresh sugarcane, at a roughage:concentrate ratio of 40:60, on the intake, dry matter (DM) and nutrient digestibility, milk yield and composition, and feeding behavior of lactating goats. Eight goats were distributed into two balanced 4 × 4 Latin squares, where the replacement levels of 0, 33, 67, and 100% were used as independent variables. Dry matter intake, which averaged 2.302 kg/day, showed a quadratic response with the minimum value observed at the sugarcane inclusion level of 68.04%. The treatments did not affect the intakes of crude protein (CP), neutral detergent fiber (NDF), or net energy, whereas non- fibrous carbohydrates (NFC) and total digestible nutrients (TDN) intakes responded quadratically and ether extract (EE) intake decreased with the increasing sugarcane levels. The levels of replacement of corn silage with sugarcane did not influence the digestibility coefficients of DM, CP, NDF, or NFC, but EE digestibility exhibited had a quadratic response. There were no significant differences for milk yield, which averaged 1.512 kg/day, even when yield was corrected for 3.5% fat; or the concentrations of protein, fat, lactose, total solids, and solids-not-fat in milk. Milk urea nitrogen, on the other hand, increased with the sugarcane levels. Fresh sugarcane can replace corn silage in the diet of low-yielding goats with a 40:60 roughage:concentrate ratio, as it will not change milk yield. Key words: crude protein, dry matter, milk production, rumen,urea 55 Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage and in sufficient quantity to allow 10% orts for sampling. Water was available ad libitum. Introduction These proportions were maintained throughout the experiment with adjustments in the amount of roughage supplied, based on their DM. Because goats are selective animals, their forage intake depends mainly on the nutritional value of the feed and their rumen-fill capacity. The feed particle size influences the feeding behavior of animals because it affects their dry matter intake as well as the feeding and rumination activities (NRC, 2001). Accordingly, the time spent on rumination is influenced by the nature of the diet and is proportional to the cell wall content of roughages. Thus, higher percentages of roughage in the diet translate into a longer rumination time (Van Soest, 1994). The experimental period was 72 days, which were divided into four 18-day periods, consisting of 12 days of adaptation and adjustment of voluntary feed intake and six days of data collection. The body weight dynamics was monitored by weighing the animals at the start of each period and at the end of the experiment, before the morning milking. Chemical analyses of the ingredients were carried out according to the methodology described in Silva and Queiroz (2002) for protein (CP), ash, ether extract (EE); and Van Soest et al. (1991) for neutral detergent fiber (NDF), acid detergent fiber (ADF), cellulose, and lignin. Total carbohydrates (TC), non-fibrous carbohydrates (NFC), and total digestible nutrients (TDN) were estimated by the equations below: In view of the above-stated facts, this study was conducted to examine the effects of replacing corn silage with fresh sugarcane at the levels of 0, 33, 67, and 100% on the intake, nutrient digestibility, performance, and feeding behavior of lactating dairy goats. TC = 100 – %CP – %EE – %Ash, according to Sniffen et al. (1992); Materials and Methods NFC = 100 – (%CP + %EE + %Ash + %NDF), according to Van Soest et al. (1991); and NFC = 100 – (%CP + %EE + %Ash + %NDF), according to Van Soest et al. (1991); and The experiment was conducted in the municipality of Botucatu - SP, Brazil (22°53'09 '' S and 48°26'42 '' W, 840 m above sea level). According to the Köppen climate classification, the region has a Cwa climate type, characterized as mild, with an average temperature of 22 °C. Eight lactating Alpine goats (post-lactation peak) with a body weight of 51.95 ± 3.29 kg were used. The animals were housed in individual 3.5-m2 stalls inside a covered shed equipped with a drinker, a salt trough, and a feed trough. The experiment was conducted in the municipality of Botucatu - SP, Brazil (22°53'09 '' S and 48°26'42 '' W, 840 m above sea level). According to the Köppen climate classification, the region has a Cwa climate type, characterized as mild, with an average temperature of 22 °C. TDN = DCP + DNFC + DNDF + DFA × 2.25 – 7, according to NRC (2001), in which: in which: in which: DCP = CP × Exp [ –1.2 × ADIP/CP] for roughage; DCP = [1 – (0.4 × ADIP/CP)] × CP for concentrate; DNFC = 0.98 × NFC; DNDF = 0.75 × (NDF – L) × [1 – (L/NDF) × 0.667]; DFA = EE – 1; and 7 represents the fecal metabolic TDN. Eight lactating Alpine goats (post-lactation peak) with a body weight of 51.95 ± 3.29 kg were used. The animals were housed in individual 3.5-m2 stalls inside a covered shed equipped with a drinker, a salt trough, and a feed trough. g g The experimental diets were supplied twice daily, at 08.00 h and 16.00 h, as a complete ration In these secondary equations, DCP represents truly digestible protein; DNFC, the truly 56 Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage digestible NFC; DNDF, the digestible NDF; DFA, the truly digestible fatty acids; ADIP, acid detergent indigestible protein; and L, acid detergent lignin. Results and discussion The voluntary intake of feed and nutrients was calculated as the difference between the supplied amount and orts. Feed and orts were collected and frozen for further analyses, which were performed after the samples were thawed, pre-dried in a forced-air oven at 55 °C for 72 h, and ground in a Wiley mill with 2-mm sieves. Feces samples were also collected to determine apparent digestibility. Dry matter intake responded quadratically to the increasing levels of corn silage replaced with sugarcane, with a minimum intake value of 2.126 kg/day recorded at the sugarcane level of 68.04%. The same was not true for DM intake evaluated as a percentage of live weight (LW). The average DM intake was 2.272 kg/day and 4.41% LW (Table 3), and the average CP intake was 0.378 kg/day (Table 4). Ether extract intake decreased linearly as the sugarcane levels replacing corn silage were increased. In the chemical analysis of orts and feces, the methodology described in Silva and Queiroz (2002) was used for CP and EE; and in Van Soest et al. (1991) for NDF and ADF. Non-fibrous carbohydrates, TDN, and NE were estimated from the previously described equations. Neutral detergent fiber intake did not differ significantly between the animals subjected to the different treatments, averaging 0.429 kg/day. The intakes of NFC and TDN differed between the animals on the different levels of corn silage replaced with sugarcane. The same was not true for net energy intake. The minimum NFC intake was 1,168 kg/day, which was recorded at 67.10% sugarcane inclusion. To calculate the digestibility of DM and nutrients from the diets, feces were collected directly from the rectum of the animals for four consecutive days in each experimental period. Fecal output was estimated using iNDF (indigestible NDF) as a marker, by 168 h of in situ ruminal incubation (Berchielli et al., 2005) of samples of feed, orts, and feces inside nylon bags with 50 μm porosity, following to the standardized technique mentioned by Vanzant et al. (1998). After incubation, the bags were washed in running water and dried in a forced- air oven at 55 °C for 72 h. Analyses of NDF were carried out as proposed by Van Soest et al. (1991), using an ANKON200 instrument (Ankom Technology Corp., Fairport, NY, USA). Materials and Methods The goats were milked twice daily, at 07.30 h and 15.30 h, and their MY was recorded for five consecutive days, in the different experimental periods. Milk samples from two milk-testing days, in the proportions of 2/3 of the morning milking and 1/3 of the afternoon milking, were collected in 30-mL plastic tubes containing the preservative bronopol (2-bromo-2-nitropropane-1, 3-diol) and sent for analysis of constituents. The milk protein, fat, lactose, total solids, solids-not-fat, and urea nitrogen contents were determined. To calculate the metabolizable energy (ME) and net energy (NE) values, the TDN and digestible energy (DE) values were used in the following equations suggested by the NRC (2001): DE (Mcal/kg) = 0.04409 × TDN (%) ME (Mcal/kg) = 1.01 × DE (Mcal/kg) – 0.45 NE (Mcal/kg) = 0.0245 × TDN (%) – 0.12 The data were subjected to analysis of variance using SAEG computer software (Statistical and Genetic Analysis System, version 9.0). Traits that showed a significant effect for treatment were studied by regression analyses, in which the effects were separated into linear, quadratic, and cubic (P<0.05). At the beginning of the data collection period, the feeding behavior of the animals was observed and recorded. Feed intake and MY were recorded and samples were collected for five consecutive days in each period. Milk was sampled on the 4th and 5th days of each collection period. Results and discussion Then, fecal output was estimated using the following equations: Total digestible nutrient intake was influenced by the levels of replacement of corn silage with sugarcane. This variable responded quadratically, with a minimum value of 1.507 kg/day recorded at 65.71% sugarcane. There was no significant effect for the digestibility of DM, CP, NDF, or NFC between the replacement levels. However, EE digestibility differed significantly (Table 3), showing a quadratic response, with a maximum coefficient of 81.04 recorded at 23.325% sugarcane. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO In the present experiment, DM intake met the energy requirements of the lactating goats (Table 3). The greater percentage of concentrate in the diets likely favored the availability of energy for the maintenance and production of the animals. Due to the preference of the animals, the sugar content of sugarcane and the starch of corn silage may have favored the increase in NFC intake in the group fed the roughage-only diet. The result for NFC intake is contrary to those reported by Magalhães et al. (2004), who found no differences in NDF intake using diets with levels of sugarcane replacing corn silage; and by Mendonça et al. (2004), who described higher NFC intake using diets with corn silage in comparison with sugarcane in the feeding of lactating cows. Several authors that worked with diets with roughage:concentrate ratios greater than 50:50 and roughages with low fiber digestibility, as is the case of sugarcane relative to corn silage, found that DM intake decreased, affecting the MY of lactating cows (Magalhães et al., 2004; Costa et al., 2005; Pires et al., 2010). In dairy goats, this ratio is also determinant for intake, since goats with greater intake capacity have greater potential for milk production (Resende et al., 2007). g g As reflected by DM intake, the higher NFC intake from the diet with corn silage relative to sugarcane may have contributed to the higher TDN intake shown by these treatment groups. The average intake of 1.56 Mcal/kg met the net energy requirement of 1,180 Mcal/kg recommended by the NRC (2007) for goats producing 1,500 kg/day of milk. This value is related to nutrient digestibility. The increase in DM intake with highly digestible nutrients favored the intake of net energy, and, consequently the increase in production. There is a negative correlation between MY and the fiber content of the diet and a positive correlation between MY and the net energy content of the forage (Morand-Fehr and Sauvant, 1980). Crude protein intake, which averaged 0.378 kg/day, was not affected by the levels of replacement of corn silage with sugarcane. The higher intake is likely due to the participation of non- protein nitrogen in the form of urea and the excess CP. However, the diets were isoproteic to meet the protein requirements of the goats, given the larger particle size of the roughage that was mixed with the concentrate daily. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO (2004), who worke containing corn silage and/or sugarc 60:40 roughage:concentrate ratio to f cows. The mean DM digestibility of higher than the 65.18% found by Vilela in diets with the inclusion of sugarca with different concentrates; and also hig 66 75% described by Costa et al (2005 of this nutrient in the chemical composition of the diets. Magalhães et al. (2004) found a linear decrease in DM intake as they increased the amount of sugarcane replacing corn silage, whereas Mendonça et al. (2004) observed higher DM intake using corn silage versus sugarcane, in the diet of lactating cows with a 60:40 roughage:concentrate ratio. These authors reported that DM intake may have been affected by the quality of roughage regardless of the feeding strategy, indicating that sugarcane has a reducing effect on intake as a consequence of the low digestibility of its fiber. The NFC content, especially sugars, and the preference for the juicier parts, may have contributed to the higher DM intake by the 100% sugarcane treatment group. The intake of NDF was also lower than the 0.710 and 0.876 kg/day found by Branco et al. (2011) and Carvalho et al. (2006), respectively, using diets with increasing levels of NDF for lactating goats. These authors reported that the increased percentage of NDF in the diet induced a decrease in DM intake, and concluded that, based on MY and the intakes of DM, fiber, and energy, the ideal NDF content is 35%. The NDF level used in the diets in the current experiment was 24.03%, which is below the values proposed by Branco et al. (2001) and Carvalho et al. (2006). Moreover, the reduction in DM intake with the sugarcane-based diets may be related to the quality of the sugarcane fiber, given its low digestibility and/or low rates of digestion and passage through the rumen (Pires et al., 2010), and not only the NDF content, as some studies showed higher NDF contents in corn silage when compared with sugarcane (Magalhães et al., 2004; Mendonça et al., 2004). g p The average DM intake of 2.272 kg/day and 4.41% LW (Table 3) is within the recommended range for goats with a LW of 50 kg, which should produce from 1.47 to 2.30 kg/day of milk (NRC, 2007). As also indicated by the council, depending on MY, dairy goats consume from 4.00 to 7.00% of their LW in DM. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO Moreover, t reduction in DM intake with the sugarcane-bas diets may be related to the quality of the sugarca fiber, given its low digestibility and/or low rates digestion and passage through the rumen (Pires al., 2010), and not only the NDF content, as som studies showed higher NDF contents in corn sila when compared with sugarcane (Magalhães et a 2004; Mendonça et al., 2004). Due to the preference of the animals, t sugar content of sugarcane and the starch of co silage may have favored the increase in NFC inta in the group fed the roughage-only diet. The res for NFC intake is contrary to those reported Magalhães et al. (2004), who found no differences NDF intake using diets with levels of sugarca replacing corn silage; and by Mendonça et (2004), who described higher NFC intake using die with corn silage in comparison with sugarcane in t feeding of lactating cows. As reflected by DM intake, the higher NF intake from the diet with corn silage relative sugarcane may have contributed to the higher TD intake shown by these treatment groups. T average intake of 1.56 Mcal/kg met the net ener requirement of 1,180 Mcal/kg recommended by t NRC (2007) for goats producing 1,500 kg/day milk. This value is related to nutrient digestibili The increase in DM intake with highly digestib nutrients favored the intake of net energy, an consequently the increase in production. There is negative correlation between MY and the fib content of the diet and a positive correlati between MY and the net energy content of t forage (Morand-Fehr and Sauvant, 1980). Results for DM and CP digestibility agr with those described by Magalhães et al. (2006) a Mendonça et al. (2004), who worked with die containing corn silage and/or sugarcane with 60:40 roughage:concentrate ratio to feed lactati cows. The mean DM digestibility of 69.16% w higher than the 65.18% found by Vilela et al. (200 in diets with the inclusion of sugarcane and ur with different concentrates; and also higher than t 66.75% described by Costa et al. (2005) in diets w corn silage or sugarcane for dairy cows. T roughage content of 40% and the similar ND contents of the experimental diets may ha Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage , pp g y, p p y g g p Magalhães et al. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO (2004) found a linear decrease in DM intake as they increased the amount of sugarcane replacing corn silage, whereas Mendonça et al. (2004) observed higher DM intake using corn silage versus sugarcane, in the diet of lactating cows with a 60:40 roughage:concentrate ratio. These authors reported that DM intake may have been affected by the quality of roughage regardless of the feeding strategy, indicating that sugarcane has a reducing effect on intake as a consequence of the low digestibility of its fiber. The NFC content, especially sugars, and the preference for the juicier parts, may have contributed to the higher DM intake by the 100% sugarcane treatment group. The average DM intake of 2.272 kg/day and 4.41% LW (Table 3) is within the recommended range for goats with a LW of 50 kg, which should produce from 1.47 to 2.30 kg/day of milk (NRC, 2007). As also indicated by the council, depending on MY, dairy goats consume from 4.00 to 7.00% of their LW in DM. In the present experiment, DM intake met the energy requirements of the lactating goats (Table 3). The greater percentage of concentrate in the diets likely favored the availability of energy for the maintenance and production of the animals. Several authors that worked with diets with roughage:concentrate ratios greater than 50:50 and roughages with low fiber digestibility, as is the case of sugarcane relative to corn silage, found that DM intake decreased, affecting the MY of lactating cows (Magalhães et al., 2004; Costa et al., 2005; Pires et al., 2010). In dairy goats, this ratio is also determinant for intake, since goats with greater intake capacity have greater potential for milk production (Resende et al., 2007). Crude protein intake, which averaged 0.378 kg/day, was not affected by the levels of replacement of corn silage with sugarcane. The higher intake is likely due to the participation of non- protein nitrogen in the form of urea and the excess CP. However, the diets were isoproteic to meet the protein requirements of the goats, given the larger particle size of the roughage that was mixed with the concentrate daily. In addition, the preference for the concentrate may have influenced the higher CP intake in the treatments. This intake value is similar to the 0.368 kg/day described by Fonseca et al. (2006) in dairy goats fed a diet with 15.5% CP. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO For EE, results were similar to those found by Magalhães et al. (2004) and Mendonça et al. (2004), who replaced corn silage with sugarcane in diets for lactating cows. The lower EE content of sugarcane favored the lower intake of this nutrient in comparison with corn silage. Results for NDF were contrary to those described by Magalhães et al. (2004) and Mendonça et al (2004) who mentioned that NDF intake of this nutrient in the chemical compo diets. The intake of NDF was also lo 0.710 and 0.876 kg/day found by B (2011) and Carvalho et al. (2006), using diets with increasing levels lactating goats. These authors repor increased percentage of NDF in the d decrease in DM intake, and concluded on MY and the intakes of DM, fiber, and ideal NDF content is 35%. The NDF the diets in the current experiment w which is below the values proposed by (2001) and Carvalho et al. (2006). M reduction in DM intake with the suga diets may be related to the quality of th fiber, given its low digestibility and/or digestion and passage through the rum al., 2010), and not only the NDF conte studies showed higher NDF contents i when compared with sugarcane (Maga 2004; Mendonça et al., 2004). Due to the preference of the sugar content of sugarcane and the s silage may have favored the increase i in the group fed the roughage-only die for NFC intake is contrary to those Magalhães et al. (2004), who found no d NDF intake using diets with levels o replacing corn silage; and by Mend (2004), who described higher NFC intak with corn silage in comparison with sug feeding of lactating cows. As reflected by DM intake, the intake from the diet with corn silag sugarcane may have contributed to the intake shown by these treatment average intake of 1.56 Mcal/kg met th requirement of 1,180 Mcal/kg recomme NRC (2007) for goats producing 1,50 milk. This value is related to nutrient The increase in DM intake with high nutrients favored the intake of net consequently the increase in productio negative correlation between MY an content of the diet and a positive between MY and the net energy co forage (Morand-Fehr and Sauvant, 1980 Results for DM and CP diges with those described by Magalhães et a Mendonça et al. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO There were no differences in MY or 3.5% fat-corrected MY between the treatment groups, despite the observed differences in DM intake. The lack of synchronization between the nitrogen sources and soluble carbohydrates to form microbial protein possibly influenced the low MY, as verified by the urea nitrogen values (Table 5), which indicate excess CP that was not used for production. Another factor to be considered, regarding the low MY, is that net energy intake was not sufficient to meet the requirements of goats with a production potential of 2.5 to 3.5 kg/day of milk (NRC, 2007). iNDFI = iNDFFd – iNDFO in which FO = fecal output (kg/day); iNDFI = iNDF intake (kg/day); iNDFFc = iNDF concentration in the feces (kg/kg); iNDFFd = iNDF intake from the feed (kg/day); and iNDFO = iNDF present in orts (kg/day). in which FO = fecal output (kg/day); iNDFI = iNDF intake (kg/day); iNDFFc = iNDF concentration in the feces (kg/kg); iNDFFd = iNDF intake from the feed (kg/day); and iNDFO = iNDF present in orts (kg/day). 57 ares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn sila al. (2004) found a linear decrease in s they increased the amount of eplacing corn silage, whereas l. (2004) observed higher DM intake ge versus sugarcane, in the diet of with a 60:40 roughage:concentrate uthors reported that DM intake may fected by the quality of roughage the feeding strategy, indicating that a reducing effect on intake as a f the low digestibility of its fiber. The especially sugars, and the preference parts, may have contributed to the ke by the 100% sugarcane treatment erage DM intake of 2.272 kg/day and able 3) is within the recommended s with a LW of 50 kg, which should 1.47 to 2.30 kg/day of milk (NRC, indicated by the council, depending oats consume from 4.00 to 7.00% of M. In the present experiment, DM energy requirements of the lactating 3). FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO The greater percentage of the diets likely favored the availability e maintenance and production of the l authors that worked with diets with entrate ratios greater than 50:50 and low fiber digestibility, as is the case elative to corn silage, found that DM ed, affecting the MY of lactating cows al., 2004; Costa et al., 2005; Pires et dairy goats, this ratio is also r intake, since goats with greater y have greater potential for milk sende et al., 2007). protein intake, which averaged 0.378 not affected by the levels of f corn silage with sugarcane. The likely due to the participation of non- n in the form of urea and the excess the diets were isoproteic to meet the ments of the goats, given the larger the roughage that was mixed with the ly. In addition, the preference for the ay have influenced the higher CP eatments. This intake value is similar kg/day described by Fonseca et al. goats fed a diet with 15.5% CP. , results were similar to those found et al. (2004) and Mendonça et al. placed corn silage with sugarcane in ing cows. The lower EE content of red the lower intake of this nutrient in h corn silage. s for NDF were contrary to those agalhães et al. (2004) and Mendonça who mentioned that NDF intake the amount of sugarcane replacing as increased. The observed NDF a consequence not only of the higher of this nutrient in the chemical composition of t diets. The intake of NDF was also lower than t 0.710 and 0.876 kg/day found by Branco et (2011) and Carvalho et al. (2006), respective using diets with increasing levels of NDF f lactating goats. These authors reported that t increased percentage of NDF in the diet induced decrease in DM intake, and concluded that, bas on MY and the intakes of DM, fiber, and energy, t ideal NDF content is 35%. The NDF level used the diets in the current experiment was 24.03% which is below the values proposed by Branco et (2001) and Carvalho et al. (2006). FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO According to the researchers, a possible explanation would be the dilution effect caused by the higher MY obtained with the treatments with corn silage. In an experiment with fresh or ensiled sugarcane in diets with a 50:50 roughage:concentrate ratio for lactating goats, Mendes (2006) found no significant differences between the treatments for MY, which averaged 1.520 kg/day. In experiments with dairy cows, Magalhães et al. (2004), Mendonça et al. (2004), and Costa et al. (2005) found no difference in the milk fat content between treatments with corn silage and/or sugarcane, having described mean values of 4.06, 3.82, and 3.44%, respectively. These authors expected a lower value for the 40% sugarcane treatment, suggesting that the increased proportion of concentrate in the diet causes a decline in acetic:propionic acid ratio and, consequently, in the fat content of milk. In dairy cows, Magalhães et al. (2004) reported that MY and 3.5% fat-corrected MY decreased linearly as the levels of substitution of corn silage with sugarcane were increased, suggesting that the quality of the sugarcane fiber limited the individual performance of the animals. Surplus ammonia in the rumen, a product of the degradation of protein and non-protein nitrogen present in urea, increases urea formation in the liver. Excess urea is then excreted in the urine and, to a lesser extent, in milk (Giaccone et al., 2007). For this reason, urea nitrogen has been used as an indicator to monitor excess CP in the diet, either in a degradable or soluble form. In a study with dairy cows, Costa et al. (2005) observed that MY was lowest when the animals received a diet with 60% sugarcane, intermediate with 50%, and highest with 40%. The latter treatment provided similar production to the diet with 60% corn silage. These authors attributed the lower MY obtained with diets with higher percentages of sugarcane to the lower DM intake, which resulted in less intake of nutrients. The urea nitrogen concentration responded linearly to the levels of replacement of corn silage with sugarcane, which was intensified with the increasing amounts of sugarcane. The preference of the goats to consume the concentrate, coupled with the higher urea content and excessive CP for the milk production level of the goats, may have contributed to this increase in the treatments that Corroborating the aforementioned, in a study with lactating cows fed diets containing increasing levels of sugarcane replacing corn silage, Pires et al. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO In the case the experiment involved diets percentage of concentrate, wh worked with increasing levels of N fat content may be considered h diets with a greater proportion which there is a greater product fatty acids, especially propionic ac may be deemed low, poss concentrated levels of fat, increasi The aforementioned findings of Pires et al. (2010), diets containing 50, 75, and provided a higher concentration o cows as compared with diets co 75% corn silage. According to t possible explanation would be caused by the higher MY o treatments with corn silage. In experiments with dairy et al. (2004), Mendonça et al. (20 al. (2005) found no difference in t between treatments with cor sugarcane, having described me 3.82, and 3.44%, respectively expected a lower value for the treatment, suggesting that the inc of concentrate in the diet cau acetic:propionic acid ratio and, co fat content of milk. Surplus ammonia in the ru the degradation of protein and no present in urea, increases urea fo Excess urea is then excreted in t lesser extent, in milk (Giaccone et reason, urea nitrogen has been us to monitor excess CP in the degradable or soluble form. The urea nitrogen concen decrease in milk was affected by the lower intakes of DM and, consequently, of energy. addition, this digestibility can be attributed to the greater proportion of NFC in the experimental diets, since, as the roughage fraction increases, DM digestibility decreases due to the increase in structural carbohydrates (Rode et al., 1985). Like bovine milk, the composition of goat milk varies with several factors, including breed, age, lactation stage, and diet. No differences were found for the CP, fat, lactose, total solids, or solid- not-fat components across the levels of replacement of corn silage with sugarcane. Magalhães et al. (2004) and Mendonça et al. (2004) observed similar results for protein, total solids, and solid-not-fat; and Costa et al. (2005) for CP, lactose, and solid-not-fat. The CP content of goat milk (3.40%) was within the range of 2.2 to 5.1% reported by Villalobos (2005). FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO The higher lipid content of the corn grains and silage forage and the consequent higher percentage of EE relative to sugarcane may have contributed to its greater digestibility, since the lipids of forage are hydrolyzed to form fatty acids, galactose, and glycerol in the rumen, the latter of which are rapidly fermented to short-chain fatty acid (Palmquist and Mattos, 2006). This greater digestibility favors the fat content of milk. g p y ( ) The average milk fat content of 3.63% was higher than the 2.8% observed by Canizares et al. (2011); 2.95% by Fonseca et al. (2006); 3.24% by Carvalho et al. (2006); and 3.32% by Branco et al. (2011) in dairy goats. In the case of the first authors, the experiment involved diets with a greater percentage of concentrate, whereas the latter worked with increasing levels of NDF in the diet. The fat content may be considered high in the case of diets with a greater proportion of concentrate, in which there is a greater production of short-chain fatty acids, especially propionic acid. The MY, which may be deemed low, possibly resulted in concentrated levels of fat, increasing its percentage. g y The average digestibility coefficients of NDF and NFC were 23.59 and 87.12%, respectively. These coefficients were lower and similar to those reported by Mendonça et al. (2004) and Costa et al. (2005), respectively, who found greater digestibility of NDF in corn silage diet as compared with the sugarcane diet (means: 39.23 vs. 43.59%). However, NFC digestibility was inversely proportional to NDF digestibility, with higher values occurring in the sugarcane diets and lower coefficients obtained with corn silage (means: 92.9 vs. 86.22%). Dry matter intake is one of the main determinants of production, as goats with greater intake capacity have greater potential for milk production (Resende et al., 2007). In this study, as there was no change in DM intake, this result can be attributed to the intake of CP. The DM intake of 2.272 kg/day was reflected in the MY of 1.512 kg/day, which is in agreement with the requirements defined by the NRC (2007) for this production level. The aforementioned corroborates the findings of Pires et al. (2010), who reported that diets containing 50, 75, and 100% sugarcane provided a higher concentration of fat in the milk of cows as compared with diets containing 100 and 75% corn silage. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO In addition, the preference for the concentrate may have influenced the higher CP intake in the treatments. This intake value is similar to the 0.368 kg/day described by Fonseca et al. (2006) in dairy goats fed a diet with 15.5% CP. Results for DM and CP digestibility agree with those described by Magalhães et al. (2006) and Mendonça et al. (2004), who worked with diets containing corn silage and/or sugarcane with a 60:40 roughage:concentrate ratio to feed lactating cows. ( ) y g For EE, results were similar to those found by Magalhães et al. (2004) and Mendonça et al. (2004), who replaced corn silage with sugarcane in diets for lactating cows. The lower EE content of sugarcane favored the lower intake of this nutrient in comparison with corn silage. The mean DM digestibility of 69.16% was higher than the 65.18% found by Vilela et al. (2003) in diets with the inclusion of sugarcane and urea with different concentrates; and also higher than the 66.75% described by Costa et al. (2005) in diets with corn silage or sugarcane for dairy cows. The roughage content of 40% and the similar NDF contents of the experimental diets may have contributed to the greater digestibility of DM. In Results for NDF were contrary to those described by Magalhães et al. (2004) and Mendonça et al. (2004), who mentioned that NDF intake decreased as the amount of sugarcane replacing corn silage was increased. The observed NDF intake may be a consequence not only of the higher percentage of concentrate, but also the similar level 58 Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage addition, this digestibility can be attributed to the greater proportion of NFC in the experimental diets, since, as the roughage fraction increases, DM digestibility decreases due to the increase in structural carbohydrates (Rode et al., 1985). The higher lipid content of the corn grains and silage forage and the consequent higher percentage of EE relative to sugarcane may have contributed to its greater digestibility, since the lipids of forage are hydrolyzed to form fatty acids, galactose, and glycerol in the rumen, the latter of which are rapidly fermented to short-chain fatty acid (Palmquist and Mattos, 2006). This greater digestibility favors the fat content of milk. The average digestibility coefficients of NDF and NFC were 23.59 and 87.12%, respectively. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO These coefficients were lower and similar to those reported by Mendonça et al. (2004) and Costa et al. (2005), respectively, who found greater digestibility of NDF in corn silage diet as compared with the sugarcane diet (means: 39.23 vs. 43.59%). However, NFC digestibility was inversely proportional to NDF digestibility, with higher values occurring in the sugarcane diets and lower coefficients obtained with corn silage (means: 92.9 vs. 86.22%). Dry matter intake is one of the main determinants of production, as goats with greater intake capacity have greater potential for milk production (Resende et al., 2007). In this study, as there was no change in DM intake, this result can be attributed to the intake of CP. The DM intake of 2.272 kg/day was reflected in the MY of 1.512 kg/day, which is in agreement with the requirements defined by the NRC (2007) for this production level. In an experiment with fresh or ensiled sugarcane in diets with a 50:50 roughage:concentrate ratio for lactating goats, Mendes (2006) found no significant differences between the treatments for MY, which averaged 1.520 kg/day. In dairy cows, Magalhães et al. (2004) reported that MY and 3.5% fat-corrected MY decreased linearly as the levels of substitution of corn silage with sugarcane were increased, suggesting that the quality of the sugarcane fiber limited the individual performance of the animals. In a study with dairy cows, Costa et al. (2005) observed that MY was lowest when the animals received a diet with 60% sugarcane, intermediate with 50%, and highest with 40%. The latter treatment provided similar production to the diet with 60% corn silage. These authors attributed the lower MY obtained with diets with higher percentages of sugarcane to the lower DM intake, which resulted in less intake of nutrients. decrease in milk was affected by t DM and, consequently, of energy. Like bovine milk, the co milk varies with several factors age, lactation stage, and diet. No found for the CP, fat, lactose, tot not-fat components across the lev of corn silage with sugarcane. (2004) and Mendonça et al. (2004 results for protein, total solids, and Costa et al. (2005) for CP, lactose The CP content of goat milk (3.40 range of 2.2 to 5.1% reported by V The average milk fat con higher than the 2.8% observed b (2011); 2.95% by Fonseca et al. Carvalho et al. (2006); and 3.32% (2011) in dairy goats. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO (2010) found higher yields using treatments with 100, 75, and 50% silage and less production with treatments containing 75 and 100% sugarcane in the roughage. For the authors, the 59 Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage involved sugarcane. The average urea nitrogen concentration of 29.85 mg/dL was high, which may be due to the greater amount of easily fermentable carbohydrates from sugarcane. These, in turn, generated the lack of synchronization with the rate of degradation of the nitrogen sources. a deficiency of degradable and soluble CP, whereas high values denote a deficiency of carbohydrates and excess CP. In the present study, the urea nitrogen values were high, especially for the treatment with 100% sugarcane (32.73 mg/dL), meaning that there was an excess of nitrogen, which is related to CP that was not used for the production of microbial protein and, consequently, for milk production. This is explained by the preference of the animals to consume the concentrate first, which contained urea in increasing amounts as the levels of sugarcane were raised. No reports have been found that allow establishing urea nitrogen values that can be considered normal in goat milk. This is not the case for cows, for which, according to Peres (2001), values considered normal are within the range of 12- 18 mg/dL. This author stated that low values indicate Table 1. Concentrates and forages chemical composition expressed in grams per kilogram of dry matter Nutrients (g/kg) Concentrates for each replacement level Forages 0% 33% 66% 100% Corn silage Sugarcane Dry matter 889 890 890 890 350 298 Organic matter 964 963 962 962 957 984 Ash 36 37 38 38 43 16 Crude Protein 204 216 230 242 76 20 Ether extract 40 39 38 38 41 16 Neutral detergent fiber 116 117 118 119 416 433 Acid detergent fiber 54 55 56 57 241 272 Total carbohydrates 689 680 669 659 841 948 Non-Fibrous Carbohydrates 572 563 551 541 424 516 Calcium 11 11 11 11 3 2 Phosphor 6 7 7 7 2 1 Total digestible nutrients 869 844 814 764 744 685 Metabolizable energy (Mcal/kg) 3.49 3.38 3.24 3.02 3.34 3.07 Net energy (Mcal/kg) 2.01 1.95 1.87 1.75 1.70 1.56 1. Concentrates and forages chemical composition expressed in grams per kilogram of dry matter Concentrates for each replacement level Forages Table 2. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO Experimental diets chemical composition Nutrients (g/kg) Replacements levels (%) 0 33 67 100 Dry matter 674 667 660 653 Organic matter 961 964 968 971 Ash 39 36 32 29 Crude protein 152 153 153 154 Ether extract 40 36 33 29 Neutral detergent fiber 236 239 242 244 Acid detergente fiber 129 133 138 143 Total carbohydrates 749 758 767 775 Non-fibrous carbohydrates 513 519 525 531 Calcium 8 8 8 8 Phosphor 5 5 4 4 Total digestible nutrients 819 796 770 732 Metabolizable energy (Mcal/kg) 3.265 3.160 3.041 2.872 Net energy (Mcal/kg) 1.887 1.830 1.766 1.675 60 Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Table 3. Dry matter and nutrient intake of lactating goats fed diets with different replacement levels of corn silage by sugarcane Variables Replacement levels (%) Means VC (%) 0 33 67 100 Dry matter (kg/d) 2.491 2.267 2.102 2.227 2.272 9.69 1Ŷ= 2.50239 – 0.0107805X + 0.0000792201X2 (R2 = 0.97; Ymin = 2.136 for X = 68.04) Dry matter (%PV) 4.723 4.457 4.177 4.289 4.412 8.98 Crude protein (kg/d) 0.397 0.369 0.355 0.389 0.378 9.79 Ether extract (kg/d) 0.100 0.086 0.074 0.071 0.083 11.21 1Ŷ= 0.097761 – 0.000301782X (r2 = 0.94) Neutral detergent fiber (kg/d) 0.468 0.427 0.376 0.442 0.429 14.23 Non-fibrous carbohydrates (kg/d) 1.355 1.231 1.155 1.219 1.240 8.84 1Ŷ= 1.35938 – 0.00569362X + 0.0000424255X2 (R2 = 0.98; Ymin = 1.168 for X = 67.10) Total digestible nutrients (kg/d) 1.757 1.544 1.529 1.566 1.599 8.58 1Ŷ= 1.74957 – 0.00738404X + 0.0000561865X2 (R2 = 0.97; Ymin = 1.507 for X = 65.71) Net energy (Mcal/kg) 1.573 1.504 1.629 1.550 1.564 9.07 VC = variation coeficient; 1P<0,05. Table 4. Apparent digestibility of dry matter and nutrients in diets with different replacement levels of corn silage by sugarcane of lactating goats Variables (%) Replacement levels (%) Means VC (%) 0 33 67 100 Dry matter 68.71 66.63 73.42 67.89 69.16 8.71 Crude protein 75.22 71.66 78.74 74.99 75.10 8.12 Ether extract 80.78 79.00 79.92 70.80 77.63 5.94 1Ŷ= 80.1509 + 0.0763989X – 0.00163828X2 (R2 = 0.88; Ymax = 81.04 para X = 23.32) Neutral detergente fiber 28.74 17.65 25.36 22.63 23.59 60.41 Acid detergente fiber 84.80 85.97 90.17 87.56 87.12 5.23 VC = variation coeficient; 1P<0,05. Table 4. References Branco, R. H.; Rodrigues, M. T.; Silva, M. M. C.; Rodrigues, C. A. F.; Queiroz, A. C. and Araújo F. L. 2011. Desempenho de cabras em lactação alimentadas com dietas com diferentes níveis de fibra oriunda de forragem com maturidade avançada. Revista Brasileira de Zootecnia, v.40, n.5, p.1061-1071. Branco, R. H.; Rodrigues, M. T.; Silva, M. M. C.; Rodrigues, C. A. F.; Queiroz, A. C. and Araújo F. L. 2011. Desempenho de cabras em lactação alimentadas com dietas com diferentes níveis de fibra oriunda de forragem com maturidade avançada. Revista Brasileira de Zootecnia, v.40, n.5, p.1061-1071. Giaccone, P.; Todaro, M. and Scatassa, M. L. 2007. Factors associated with milk urea concentrations in Girgentana goats. Italian Journal of Animal Science, v.6, n.1, p.622-624. Berchielli, T. T.; Oliveira, S. G.; Carilho, E. N. V. M.; Feitosa, J. V. and Lopes, A. D. 2005. Comparação de marcadores para estimativa de produção fecal e de fluxo de digesta em bovinos. Revista Brasileira de Zootecnia, v.34, n.3, p.987-996. Magalhães, A. L. R.; Campos, J. M. S.; Valadares Filho, S. C.; Torres, R. A.; Neto, J. M. and Assis, A. J. 2004. Cana-de-açúcar em substituição à silagem de milho em dietas para vacas em lactação: Desempenho e viabilidade económica. Revista Brasileira de Zootecnia, v.33, n.5, p.1292-1302: doi:10.1590/S1516-35982004000500022. Campos, P. R. S. S.; Valadares Filho, S. C.; Detmann, E. and Cecon, P. R. Consumo, digestibilidade e estimativa do valor energético de alguns volumosos por meio da composição química. 2010. Revista Ceres, v.57, n.1, p.79-86. Mendes, C. Q. 2006. Silagem de cana-de-açúcar na alimentação de ovinos e caprinos: valor nutritivo, desempenho e comportamento ingestivo. 2006. 103p. Dissertação (Mestrado em Ciência Animal e Pastagens) - Escola Superior de Agricultura "Luiz de Queiroz", Universidade de São Paulo, Piracicaba. Canizares, G. I. L.; Gonçalves, H. C.; Costa, C.; Rodrigues, L; Meneses, J. J. L.; Gomes, H. F. B.; Marques, R. O. and Branco, R. H. 2011. Use of high moisture corn silage replacing dry corn on intake, apparent. Revista Brasileira de Zootecnia, v.40, n.4, p.860-865. Mendonça, S. S.; Campos, J. M. S.; Valadares Filho, S. C.; Valadares, R. F. D.; Soares, C. A.; Lana, R. P.; Queiroz, A. C.; Assis, A. J. and Pereira, M. L. A. 2004. Consumo, digestibilidade aparente, produção e composição do leite e variáveis ruminais em vacas leiteiras alimentadas com dietas à base de cana-de-açúcar. Revista Brasileira de Zootecnia, v.33, n.2, p.481-492. https://doi.org/10.1590/S1516- 35982004000200027. FO = iNDFI/iNDFFc and iNDFI = iNDFFd – iNDFO Apparent digestibility of dry matter and nutrients in diets with different replacement levels of corn silage by sugarcane of lactating goats ent digestibility of dry matter and nutrients in diets with different replacement levels of corn silage by tating goats Table 4. Apparent digestibility of dry matter and nutrients in diets with different replacement levels o sugarcane of lactating goats 61 Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Cañizares et al. Intake, apparent digestibility, and productive parameters of dairy goats fed sugarcane replacing corn silage Table 5. Production and milk composition from lactating goats fed diets with different replacement levels of corn silage by sugarcane Variables Replacement levels (%) Means VC (%) 0 33 67 100 Milk production (kg/d) 1.670 1.441 1.474 1.465 1.512 18.03 Milk Production 3,5% fat (kg/d) 1.741 1.528 1.464 1.463 1.549 16.35 Proteina (g/kg) 32.3 34.4 34.7 34.4 34.0 6.93 Fat (g/kg) 36.4 37.6 36.3 34.7 36.3 9.85 Lactose (g/kg) 43.5 44.5 44.0 44.2 44.0 3.33 Total solids (g/kg) 120.5 125.0 123.2 120.4 122.3 4.49 Degreased dry extract (g/kg) 84.1 87.3 86.8 85.7 86.0 3.12 Urea nitrogen (mg/dL) 26.84 28.84 31.01 32.73 29.85 11.71 1Ŷ= 26.8936 + 0.0595189X (R2 = 0.99) VC = Variation coeficient; 1P<0,05. D. P. and Teixeira, M. P. Desempenho produtivo de vacas leiteiras alimentadas com diferentes proporções de cana-de-açúcar e concentrado ou silagem de milho na dieta. Revista Brasileira de Zootecnia, v.34, n.6, p.2437-2445, 2005. https://doi.org/10.1590/S1516-35982005000700032. Conclusion Sugarcane can replace 100% of corn silage, at a 40:60 roughage:concentrate ratio, in the diet of dairy goats without changing dry matter digestibility, milk yield, or the milk protein, fat, lactose, and total solid contents. However, sugarcane inclusion increases the urea nitrogen concentration in the milk of lactating goats. Fonseca, C. E. M. D.; Valadares, R. F. D.; Valadares Filho, S. D. C.; Rodrigues, M. T.; Marcondes, M. I.; Porto, M. O.; Pina, D. S. and Moraes, K. A. 2006. Produção de leite em cabras alimentadas com diferentes níveis de proteína na dieta: consumo e digestibilidade dos nutrientes. Revista Brasileira de Zootecnia, v.35, n.3, p.1162- 1168. Fonseca, C. E. M. D.; Valadares, R. F. D.; Valadares Filho, S. D. C.; Rodrigues, M. T.; Marcondes, M. I.; Porto, M. O.; Pina, D. S. and Moraes, K. A. 2006. Produção de leite em cabras alimentadas com diferentes níveis de proteína na dieta: consumo e digestibilidade dos nutrientes. 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https://openalex.org/W4381943628	https://clinicalproteomicsjournal.biomedcentral.com/counter/pdf/10.1186/s12014-023-09413-0	English	null	Identification of ORM1, vWF, SPARC, and PPBP as immune-related proteins involved in immune thrombocytopenia by quantitative LC-MS/MS	Clinical proteomics	2,023	cc-by	7,957	© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Abstract Background Immune thrombocytopenia (ITP) is a common autoimmune disease characterized by loss of immune tolerance to platelet autoantigens leading to excessive destruction and insufficient production of platelets. Method Quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed to detect the differentially expressed proteins in bone marrow samples from active ITP patients and normal controls. Result Our bioinformatic analysis identified two upregulated proteins (ORM1 and vWF) and two downregulated proteins (PPBP and SPARC) related to immune function. The four proteins were all found to be related to the tumor necrosis factor (TNF) -α signalling pathway and involved in the pathogenesis of ITP in KEGG pathway analysis. Conclusion Bioinformatics analysis identified differentially expressed proteins in bone marrow that are involved in the TNF-α signalling pathway and are related to the activation of immune function in ITP patients. These findings could provide new ideas for research on the loss of immune tolerance in ITP patients. Keywords Immune thrombocytopenia, LC-MS/MS, TNF-α Introduction †Dong-mei Yin and Dai Yuan contributed equally to this article. Identification of ORM1, vWF, SPARC, and PPBP as immune-related proteins involved in immune thrombocytopenia by quantitative LC-MS/MS Dong-mei Yin1,2†, Dai Yuan3,4†, Rui-jie Sun5, Hong-zhi Xu3,4, Shou-yong Hun1,2, Xiao-hui Sui3,4 and Ning-ning Shan3,4* †Dong-mei Yin and Dai Yuan contributed equally to this article. Immune thrombocytopenia (ITP) is an acquired auto immune disease that is characterized by increased destruction and decreased formation of platelets due to autoimmune dysregulation [1]. The mechanism of ITP mainly involves loss of immune tolerance. Platelet membrane glycoproteins (GPs), especially GPIIb/IIIa and GPIb/IX, are recognized by autoreactive T cells and thus can activate B cells to produce anti-platelet anti bodies. These anti-platelet antibodies can then bind to the platelet membrane antigen, leading to elimination of sensitized platelets by the monocyte-macrophage sys tem [2–5]. Dysregulation of T cell activity and abnormal 1Department of Blood Transfusion, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250021, China 2Department of Blood Transfusion, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, Shandong 250021, China 3Department of Hematology, Shandong Provincial Hospital, Shandong University, Jinan, Shandong 250021, China 4Department of Hematology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, 324 Jing Wu Rd, Jinan 250021, Shandong, China 5 5Department of Rheumatology, Clinical Immunology Center, Peking Union Medical College Hospital, Beijing 100000, China Clinical Proteomics Clinical Proteomics Yin et al. Clinical Proteomics (2023) 20:24 https://doi.org/10.1186/s12014-023-09413-0 Open Access Patients and control subjectsh The bone marrow was collected from twenty newly diag nosed active ITP patients (12 females and 8 males; age range: 18-70years; median age: 42years) and twenty nor mal controls (12 females and 8 males; age range, 18–55 years; median age, 46 years) at the Department Hematol ogy of Shandong Provincial Hospital during January 2019 to December 2019. All patients fit the newly published criteria and none of them had been treated with gluco corticoids prior to sampling [12]. The platelet counts were from 1× 109/L to 30× 109 /L, with a median count of 11× 109/L (Table 1). Patients with diabetes, hyper tension, cardiovascular diseases, pregnancy, active or chronic infections, or connective tissue diseases such as systemic lupus erythematosus (SLE) were excluded. We determined the number of samples according to the actual number of ITP patients fit the criteria above dur ing January 2019 to December 2019. Patients and con trols samples were divided into four groups respectively based the similar age and sex composition to reduce the differences between groups and increase the accuracy of the results. However, there were no statistical difference in the sex, age and platelet counts between patient groups and normal groups after statistical analysis. The samples were collected with heparin anticoagulant tube and then the mononuclear cells were separated with lymphocyte separation solution and stored in – 80℃ freezers for the future test. Megakaryocytes are mature cells that originate from haematopoietic stem cells and produce platelets in bone marrow [8]. Megakaryopoiesis is a complex process that involves the complete differentiation of megakaryocytes progenitors into functional platelets and occurs specifi cally in bone marrow. Impaired maturation of megakary ocytes has been proven to be one of the mechanisms of ITP [9]. Thus, bone marrow is an important medium for ITP research. Proteomics has become critical for biological research and is recognized as a key method for novel biomarker discovery and personalized therapy development [10]. Parallel reaction monitoring (PRM) is a kind of targeted quantitative proteomics with better specificity, sensitiv ity and repeatability which can quantify multiple target proteins in complex samples at the same time. The PRM- based targeted method is an efficient approach for sam ples that are available in limited amounts due to various reasons [11]. So PRM assay has emerged as an alternative targeted method of quantification. Materials and methods expression of cytokines have been suggested to be involved in immune intolerance in ITP [6, 7]. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Yin et al. Clinical Proteomics (2023) 20:24 Page 2 of 11 Yin et al. Clinical Proteomics (2023) 20:24 Page 2 of 11 Yin et al. Clinical Proteomics PT = Petechiae, EC = ecchymoses, EP = epistaxis, GUH = genitourinary hemorrhage, GH = gingival hemorrhage Patients and control subjectsh In this study, we used quantitative liquid chromatography tandem mass spec trometry (LC-MS/MS) analysis to identify differentially expressed immune-related proteins in bone marrow. Parallel reaction monitoring (PRM) mass spectrometry was used to quantify the target proteins between ITP and control samples with the aim of identifying a new strat egy for ITP research. Informed consent was obtained from each participat ing patient and/or legal guardian. The ethical protocol followed guidelines of the research code and the research Table 1 Clinical characteristics of ITP patients Group 1 Sex/Age (year) F/18 M/27 F/41 M/45 F/65 Bleeding symptoms EC NONE PT, GUH EC, GH EP, GH Platelet counts (× 109/L) 30 18 17 8 29 Group 2 Sex/Age (year) F/18 M/33 F/48 F/43 M/60 Bleeding symptoms PT GH GH EP, GH EC, GH Platelet counts (× 109/L) 11 11 12 4 4 Group3 Sex/Age (year) F/19 F/38 F/43 M/55 M/70 Bleeding symptoms PT, EC PT, GUH EP, GH GH NONE Platelet counts (× 109/L) 9 7 12 3 14 Group 4 Sex/Age (year) M/25 M/39 F/43 F/48 F/69 Bleeding symptoms GH, EP GH PT, GH PT, GH PT, GUH Platelet counts (× 109/L) 12 14 1 3 8 Median (min–max) Age (year) 42 (18–70) Platelet counts (× 109/L) 11 (1–30) PT = Petechiae, EC = ecchymoses, EP = epistaxis, GUH = genitourinary hemorrhage, GH = gingival hemorrhage Table 1 Clinical characteristics of ITP patients (2023) 20:24 Yin et al. Clinical Proteomics (2023) 20:24 Page 3 of 11 Page 3 of 11 Yin et al. Clinical Proteomics were searched against human Uniprot database concat enated with reverse decoy database. Trypsin/P was speci fied as cleavage enzyme allowing up to 2 missing cleavages. The minimum peptide length was set to 7 amino acid residues and the maximum number of modi fications was set to 5. The mass tolerance for precursor ions was set as 20 ppm in First search and 5 ppm in Main search, and the mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as fixed modification and acetylation modification and oxi dation on Met were specified as variable modifications. FDR was adjusted to < 1% and minimum score for modi fied peptides was set > 40. ethics committee of Shandong Provincial Hospital Affili ated to Shandong University and Shandong Provincial Hospital Affiliated to Shandong First Medical University. Protein extraction and trypsin digestion Sample was sonicated three times on ice using a high intensity ultrasonic processor in lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail), Then the protein extrac tion and the trypsin digestion were conducted according to the previous study [13, 14]. Briefly, the samples were centrifuged at 12,000 g for 10 min at 4° C to remove cell debris and the supernatant was collected to a new centri fuge tube. After removing the high abundant protein by following the Pierce™ Top 12 Abundant Protein Deple tion Spin Columns kit (Thermo, Waltham, USA), the concentration determination was tested with BCA kit (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s instructions. The protein solution was reduced with 5 mM dithiothreitol (Sigma-Aldrich, Saint Louis, USA) for 30 min at 56 °C and alkylated with 11 mM iodoacetamide (Sigma-Aldrich, Saint Louis, USA) for 15 min at room temperature in darkness. Next, the urea concentration of the sample was diluted to less than 2 M. The protein solution was digested with trypsin (Pro mega, Madison, USA) at a trypsin/protein ratio of 1:50 (w/w) overnight and at a trypsin/protein ratio of 1:100 (w/w) for an additional 4 h-digestion. Bioinformatics analysis y Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database(http://www.ebi.ac.uk/ GOA/) [15]. InterProScan, an algorithm software based on protein sequence, predicted the GO function of the protein in the case of certain identified proteins without annotations in Uniprot-GOA and then classified the pro teins according to cellular composition, molecular func tion, or biological process. The protein domain functional description identified in this study were annotated by InterProScan (http://www.ebi.ac.uk/interpro/). We used KEGG pathway database (v.2.0, http://www.genome.jp/ Parallel reaction monitoring (PRM) analysishi The quantitative identification of immune-related pro teins was processed using targeted proteome quantifi cation technique based on mass spectrometry-parallel reaction monitoring (PRM). The proteins used for PRM validation were based on the results of quantitative pro teomic analysis of bone marrow serum above. The pro cedure of protein extraction and trypsin digestion was similar to those mentioned above. After being separated by ESAY-NLC 1000 UPLC system (Thermo, USA), the peptides were implanted into NSI ion source for ion ization and tandem mass spectrometry analysis by Q Exactive TM plus. The PRM data were processed using Skyline (v.3.6) software. The electrospray voltage applied was 2.0 kV. The m/z scan range was 365 to 970 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting as 27 and the fragments were detected in the Orbitrap at a resolution of 17,500. Auto matic gain control (AGC) was set at 3E6 for full MS and 1E5 for MS/MS. The maximum IT was set at 160 ms for full MS and auto for MS/MS. The isolation window for MS/MS was set at 1.6 m/z. The transitions in the PRM list were as followed: precursor charges were set as 2, 3, ion charges were set as 1, 2, ion types were set as b, y. The product ions were set as from ion 3 to last ion, the ion match tolerance was set as 0.02 Da. HPLC fractionation and LC-MS/MS analysish The peptides were fractionated by high-pH reverse HPLC with the chromatographic column Agilent 1260 (5 µ m particle size, 4.6 mm inner diameter, 250 mm length) (Agilent, USA). After being dissolved in solvent A [0.1% formic acid (Sigma) in 2% acetonitrile (ThermoFisher), the peptides were separated by EASY-nLC 1000 UPLC system (Thermo, USA). The gradient elution was similar to our previous study [13, 14]. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exac tive™ Plus (Thermo, Germany) coupled online to the UPLC. The electrospray voltage applied was 2.4 kV. The m/z scan range was 385 to 1500 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 60,000. Peptides were then selected for MS/MS using NCE setting as 30 and the fragments were detected in the Orbitrap at a resolution of 15,000. A data-dependent pro cedure (DDA) that alternated between one MS scan fol lowed by 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. Fixed first mass was set as 100 m/z. Database searchingh The resulting MS/MS data was proceeded with Max Quant search engine (v.1.5.2.8). Tandem mass spectra Page 4 of 11 Page 4 of 11 Yin et al. Clinical Proteomics (2023) 20:24 Yin et al. Clinical Proteomics show the enrichment of the differentially expressed pro tein against all identified proteins. A P-value less than 0.05 was considered statistically significant.hf kaas-bin/kaas_main) to annotate the protein pathway and then matched into the corresponding pathways in the database by KEGG mapper. Wolfpsort (v.0.2, http:// www.genscript.com/psort/wolf_psort.html)was used to annotate the subcellular localization of the protein [16]. For functional enrichment, including the enrichment of GO analysis, protein domain and KEGG pathway, a two- tailed Fisher’s exact test was used to test the enrichment of the differentially expressed protein in the all identi fied proteins, and the analysis with a P-value < 0.05 was considered significant. The clustering relationship was visualized by using the Heatmap.2 function from the R language ‘gplots’ package (v.2.0.3, https://cran.r-project. org/web/packages/cluster/). All differentially expressed protein database accession or sequence were searched against the STRING database version 10.5 for protein- protein interactions [17]. Only interactions between the proteins belonging to the searched data set were selected, thereby excluding external candidates. STRING defines a metric called “confidence score” to define interaction confidence; we fetched all interactions that had a confi dence score ≥ 0.7 (high confidence). Interaction network form STRING was visualized in R package “networkD3”. y gi The biological processes enrichment of the differentially expressed proteins showed that the upregulated proteins were mainly enriched in the acute-phase response, acute inflammatory response, regulation of interleukin (IL)-6 production and regulation of leukocyte-mediated immu nity terms, with which ORM1 was associated, as well as the blood coagulation and fibrin clot formation terms, with which vWF was associated (Fig. 1a). The down regulated proteins were mainly enriched in the regula tion of cell morphogenesis term, with which SPARC was associated, and in the secretion by cell term, with which PPBP was associated (Fig. 1b). In the molecular function category, the upregulated proteins were enriched in the binding and protein binding terms, which were related to the immune-related proteins ORM1 and vWF, and in the glycoprotein binding term, which was related to vWF (Fig. 1c). Database searchingh In the downregulated proteins analysis, a cer tain number of proteins were enriched in the cytoskeletal protein binding and enzyme binding terms; terms related to immunity, protein complex binding and macromo lecular complex binding were all associated with SPARC (Fig. 1d). Functional enrichment of immune-related proteinsii To understand the identified proteins and quantified pro teins in the database searching process, we annotated the proteins in details from gene ontology (GO), protein domain and KEGG pathway. After annotation, we further performed a GO, KEGG pathway, and protein domain enrichment analysis with the intention of assessing whether there was a significant protein enrichment trend in some functional categories. A bubble chart obtained by the two-tailed Fisher’s exact test was employed to Overview of protein identification The KEGG-based functional enrichment analysis showed that the upregulated proteins were enriched in complement and coagulation cascades, in which vWF participates. vWF was also involved in the PI3K-Akt signalling pathway, focal adhesion and ECM-receptor interaction (Supplementary Table S1), which are associ ated with the TNF-α signalling pathway (Supplementary Fig. 1). While the downregulated proteins were enriched in the carbon metabolism, biosynthesis of amino acids and glycolysis/gluconeogenesis pathways. There were other enriched KEGG functional pathways. For example, the cytokine-cytokine receptor interactions and che mokine signalling pathway which were related to PPBP maybe associated with the immune function in ITP. (Sup plementary Table S1). In this study, altogether 829 proteins were identified based on 5130 unique peptides in human bone marrow mononuclear cells (BMMCs). The confident identifica tion of proteins required an FDR < 0.01, therefore, 613 proteins met the above criteria. To check the acquired MS data, we verified that the mass error was between – 5 and 5 ppm, which meets the requirement of mass accuracy. Most peptides ranged from 7 to 20 amino acids in length, which is consistent with the basic prin ciple of trypsin digestion The cutoff for the identifi cation of proteins or differentially expressed proteins was a P value < 0.05. A greater than 1.5-fold change was termed upregulation, while a fold change of < 1/1.5 was termed downregulation for proteins. Of the differentially expressed proteins between ITP patients and controls, 26 were upregulated and 69 were downregulated including ORM1, vWF, PPBP and SPARC. Furthermore, the domain enrichment of the upregu lated proteins was enriched in lipocalin/cytosolic fatty- acid binding domain, calycin domain and calycin-like domain, which were associated with the immune-related protein ORM1 (Fig. 1e). In the downregulated protein analysis, a certain number of proteins were enriched in thioredoxin-like fold. Other CXC chemokine domain and chemokine IL-8-like domain existed in PPBP (Fig. 1f). Discussion ITP is an autoimmune disease, and loss of immune tol erance participates in its pathogenesis. In this study, we performed quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis to identify dif ferentially expressed proteins in ITP patients and normal controls. We found two upregulated proteins, ORM1 and vWF, and two downregulated proteins, PPBP and SPARC, associated with immune system processes by quantitative LC-MS/MS. We traced the four proteins according to proteins annotation, KEGG pathway and cluster analysis and found the four proteins were all related to the TNF-α signalling pathway, which of importance in the pathogen esis in ITP.l i In the cluster analysis for the biological process cate gory, the upregulated proteins (Q3 and Q4) were highly enriched in the defense response, regulation of IL-6 production, acute-phase response, acute inflammatory response, immune effector process, negative regulation of cytokine production and leukocyte mediated immu nity terms, all of which were associated with ORM1. In the downregulation group, the proteins were mainly enriched in secretion, secretion by cell and regulation of cell morphogenesis, positive regulation of transmem brane, regulation of integrin activation, while the secre tion by cell and regulation of cell morphogenesis terms were associated with PPBP and SPARC (Fig. 2a). In the molecular function category, the proteins were highly enriched Q1 and Q2, including CXCR chemokine recep tor binding, protein complex binding, macromolecular complex binding, which were associated with SPARC and PPBP. The upregulated proteins of cluster analysis, the proteins were enriched in the binding and protein bind ing terms, which were associated with vWF and ORM1, and in the chaperone binding term, which was associated with vWF (Fig. 2b). In the cellular component category, the enriched proteins were mostly downregulated pro teins. Regarding immune function, only the extracellular space term was enriched, which was related to ORM1 among the upregulated proteins (Fig. 2c). TNF-α is an important inflammatory cytokine whose levels are increased in some autoimmune diseases, such as SLE, rheumatoid arthritis, and inflammatory bowel disease [18–20]. Increased levels of TNF-α have also been found in ITP patient serum [21, 22]. Treatment using blockers of TNF-α has been found to increase the number of platelets in refractory ITP patients, indicat ing that TNF-α may be an important participant in the pathogenesis of ITP [23]. TNF-α binding to the TNF-α receptor induces a series of signal transduction pathways and thus regulates cell growth, differentiation, apoptosis and inflammation. Cluster analysis Based on the analysis of proteins in the different com parison groups for the GO classification, KEGG pathway and protein domain enrichment, we applied cluster anal ysis in the comparison groups to assess the correlations Yin et al. Clinical Proteomics (2023) 20:24 Page 5 of 11 Page 5 of 11 (2023) 20:24 Yin et al. Clinical Proteomics ig. 1 Fisher’s exact test was used for functional classification and to obtain the pathways with significant protein enrichment (P < 0.05) A: The biologic rocess enriched in upregulated proteins B: Enrichment analysis of the biological processes of the downregulated proteins. C: Enrichment analysis of th molecular functions of the upregulated proteins. D: Enrichment analysis of the molecular functions of the downregulated proteins E: Domain enrichme nalysis of the upregulated proteins. F: Domain enrichment analysis of the downregulated proteins Fig. 1 Fisher’s exact test was used for functional classification and to obtain the pathways with significant protein enrichment (P < 0.05) A: The biological process enriched in upregulated proteins B: Enrichment analysis of the biological processes of the downregulated proteins. C: Enrichment analysis of the molecular functions of the upregulated proteins. D: Enrichment analysis of the molecular functions of the downregulated proteins E: Domain enrichment analysis of the upregulated proteins. F: Domain enrichment analysis of the downregulated proteins Page 6 of 11 Page 6 of 11 Yin et al. Clinical Proteomics (2023) 20:24 Yin et al. Clinical Proteomics (2023) 20:24 Yin et al. Clinical Proteomics between the functions of differentially expressed pro teins. All the categories obtained in the enrichment analysis along with their P values were first collated for further hierarchical clustering based on the functional classifications of the differentially expressed proteins. Then, the categories that were enriched in at least one cluster with a P value < 0.05 were filtered. We divided the differentially expressed proteins into four parts accord ing to their fold changes, quartile (Q) 1 through Q4. Spe cifically, the proteins were divided according to their P/C ratios (Q1, 0 < P/C ratio < 1/2; Q2, 1/2 < P/C ratio < 1/1.5; Q3, 1.5 < P/C ratio < 2; and Q4, P/C ratio > 2), as shown in the figure. The upregulated proteins were classified into Q3 and Q4, while the downregulated proteins were clas sified into Q1 and Q2. Cluster analysis Protein-protein interaction network Protein-protein interaction network To show the protein-protein interactions clearly, we selected the top 50 proteins with the closest interaction relationships and drew a protein-protein interaction net work using the STRING (V.10.5) database. The immune- related proteins ORM1 and vWF were upregulated in the interaction network, while SPARC and PPBP were downregulated. The protein-protein network is depicted in Fig. 3a and the protein levels of ORM1 and vWF were clearly higher in ITP patients than in controls, while the protein levels of SPARC and PPBP were obviously lower in ITP patients than in controls (Fig. 3b). Discussion Nuclear factor kappa B (NF-κB), mito gen-activated protein kinase (MAPK) and c-Jun N-termi nal kinase (JNK) are important signalling pathways in the downstream regulatory pathway of TNF-α in the mono cyte macrophage system [24].f In the KEGG pathway cluster analysis, the proteins were mostly downregulated proteins; among the upregu lated proteins, the proteins were enriched in complement and coagulation cascades, which were associated with vWF (Fig. 2d).h In the biological processes enrichment of the differ entially expressed proteins, the upregulated proteins were mainly enriched in the acute-phase response, acute inflammatory response, regulation of interleukin (IL)-6 production and regulation of leukocyte-mediated immunity terms, with which ORM1 was associated. ORM1 was found ORM1, also called α-1-acid glyco protein (AGP1), is a glycoprotein with a low isoelectric point (pI) of 2.8–3.8 and a high carbohydrate content of 45% [25]. The biological function of ORM1 is related to its immunomodulatory properties and its ability to bind The domain category-based enrichment analysis indi cated that the proteins were mostly enriched in downreg ulation groups. The chemokine interleukin-8-like domain and CXC chemokine domain were both associated with PPBP. Among the upregulated proteins, the calycin-like domain, lipocalin/cytosolic fatty-acid binding domain and calycin domain were all associated with ORM1 (Fig. 2e). Page 7 of 11 Yin et al. Clinical Proteomics (2023) 20:24 drugs [26 27] ORM1 is an acute phase protein whose with chronic active antibody mediated rejection than in Fig. 2 Cluster analysis was performed to determine the correlations between the functions of the differentially expressed proteins in the comparison groups. A: The cluster analysis of biological processes. B: The molecular function category analysis. C: The cellular component analysis. D: The KEGG path way analysis. E: The protein domain analysis Yin et al. Clinical Proteomics (2023) 20:24 Page 7 of 11 Yin et al. Clinical Proteomics Fig. 2 Cluster analysis was performed to determine the correlations between the functions of the differentially expressed proteins in the comparison groups. A: The cluster analysis of biological processes. B: The molecular function category analysis. C: The cellular component analysis. D: The KEGG path way analysis. E: The protein domain analysis Fig. 2 Cluster analysis was performed to determine the correlations between the functions of the differentially expressed proteins in the comparison groups. A: The cluster analysis of biological processes. B: The molecular function category analysis. C: The cellular component analysis. D: The KEGG path way analysis. Discussion E: The protein domain analysis drugs [26, 27]. ORM1 is an acute-phase protein whose levels increase rapidly under conditions of inflamma tion, stress, chronic diseases (cancer, etc.) and injury and can be modulated by glucocorticoids, TNF-α, IL-1, IL-8, IL-6, and IL-6-related cytokines [28, 29]. Komori confirmed that ORM1 can concentration-dependently increase TNF-α, IL-6, and IL-10 levels above normal serum levels, plateauing at an acute-phase concentration, and found that phosphorylation of NF-κB, p38 and JNK occurs after incubation with ORM1 [30]. Higuchi con firmed that ORM1 can enhance NF-κB-mediated inflam mation in vivo and found that ORM1 levels are higher in kidney transplant recipients pathologically diagnosed drugs [26, 27]. ORM1 is an acute-phase protein whose levels increase rapidly under conditions of inflamma tion, stress, chronic diseases (cancer, etc.) and injury and can be modulated by glucocorticoids, TNF-α, IL-1, IL-8, IL-6, and IL-6-related cytokines [28, 29]. Komori confirmed that ORM1 can concentration-dependently increase TNF-α, IL-6, and IL-10 levels above normal serum levels, plateauing at an acute-phase concentration, and found that phosphorylation of NF-κB, p38 and JNK occurs after incubation with ORM1 [30]. Higuchi con firmed that ORM1 can enhance NF-κB-mediated inflam mation in vivo and found that ORM1 levels are higher in kidney transplant recipients pathologically diagnosed with chronic active antibody-mediated rejection than in kidney transplant recipients with normal histology [31]. These findings indicate the important role of ORM1 in immune disease. Therefore, in our study, the increase in ORM1 could have induced the expression of TNF-α directly or via activation of the NF-κB, p38 and JNK pathways, which are downstream of the TNF-α signal ling pathway, to induce an immunological effect. vWF, one of the most common glycoproteins in the processes of thrombosis and haemostasis, is also a well-known index of endothelial damage and is involved in mediat ing the adhesion of platelets to subendothelial tissues [32]. In addition, vWF has been found to be involved in Page 8 of 11 Page 8 of 11 (2023) 20:24 Yin et al. Clinical Proteomics (2023) 20:24 Yin et al. Clinical Proteomics Fig. 3 A: Protein-protein interactions of the differentially expressed proteins. The red bubbles represent the immune-related proteins ORM1, vWF, SPARC and PPBP; these four proteins interact with each other. The blue bubbles represent other differentially expressed proteins. B: Mass spectrometry-based targeted proteomics was conducted to quantify the four immune-related proteins. Discussion The levels of ORM1 and vWF were significantly higher in ITP samples than in control samples, while the levels of SPARC and PPBP were lower in ITP samples than in control samples. Two-tailed Student’s t-tests were used to compare the two groups. P < 0.05, P < 0.01, *P < 0.001 Fig. 3 A: Protein-protein interactions of the differentially expressed proteins. The red bubbles represent the immune-related proteins ORM1, vWF, SPARC and PPBP; these four proteins interact with each other. The blue bubbles represent other differentially expressed proteins. B: Mass spectrometry-based targeted proteomics was conducted to quantify the four immune-related proteins. The levels of ORM1 and vWF were significantly higher in ITP samples than in control samples, while the levels of SPARC and PPBP were lower in ITP samples than in control samples. Two-tailed Student’s t-tests were used to compare the two groups. P < 0.05, P < 0.01, *P < 0.001 by phosphorylating Smad2 [38, 39]. TGF-β is a pleio tropic cytokine that regulates a broad range of cellular processes, such as differentiation, proliferation, migra tion, survival and apoptosis, and is downregulated in ITP patients [2, 40]. In the TGF-β signalling pathway, Smad2/3 can be suppressed by Smad6/7, which can be indirectly modulated by TNF-α [9]. However, SPARC deficiency in leukocytes results in exacerbation of inflam mation owing to impaired TGF-β1-mediated TNF-α downregulation [41]. Taken together, these findings indi cate that SPARC may be associated with cross-regulation of TNF-α and TGF-β in inflammatory infiltration. The downregulation of SPARC synthesis by inflammatory cytokines, including IL-1 and TNF-α, during the acute phase of arthritis, as verified by Nakamura, further sup ports our hypothesis about the role of SPARC in immune function and suggests that TNF-α can modulate SPARC reversal [42]. In our study, the downregulation of SPARC was consistent with the hypothesis that SPARC downreg ulation decreased TGF-β. In the cluster analysis of pro tein domains, the chemokine IL-8-like domain and the CXC chemokine domain were both associated with PPBP, which is also called CXC chemokine ligand 7 (CXCL7), and NAP-2, a platelet-derived growth factor belonging to the CXC chemokine family [43]. CXCL7 (PPBP) plays a prominent role in recruiting neutrophils to the injury site during thrombosis [44]. Dysregulation of CXCL7 has been implicated in inflammatory diseases, such as RA, acute lung injury and chronic obstructive pulmonary disease [45–47]. In addition, CXCL7 has been identified inflammation [33]. Discussion In addition, Chiang found that CXCL7 expression can be inhibited by IL-6 [51], a downstream cytokine in the TNF-α signalling pathway, which is one of the mechanisms responsible for activating the host immune system against tumors. Therefore, we speculate that PPBP associates with the TNF-α signalling pathway via IL-6 and TNF-α downstream and plays an immuno logical role in Th2 polarization. In this study, PPBP was found to be downregulated; thus, it could weaken the polarization of Th2 cells, which is consistent with the findings of a previous study on Th1 polarization in ITP [52]. revealed the key role of immunoregulation in the patho genesis of ITP and the significant roles of the upregulated proteins ORM1 and vWF and the downregulated pro teins SPARC and PPBP in the immunological function of ITP via LC-MS/MS. Our findings are of great signifi cance for the diagnosis and treatment of ITP patients in the future. In the follow-up experiment, Western Blot, PCR, ELISA and vivo experiments would be conducted to verify the expression levels of ORM1, vWF, SPARC and PPBP. The function role of the four proteins would be detected by enhancing or blocking the proteins in TNF-α signalling pathway in ITP mouse models to find out the role of four proteins in the pathogenesis in ITP. Abbreviations ITP immune thrombocytopenia ORM1 orosomucoid 1 vWF von Willebrand factor PPBP pro-platelet basic protein SPARC secreted protein acidic and rich in cysteine GPs glycoproteins MKs megakaryocytes IL interleukin LC-MS/MS liquid chromatography tandem mass spectrometry PRM parallel reaction monitoring TNF-α tumor necrosis factor-α TGF-β transforming growth factor -β NF-κB nuclear factor kappa beta MAPK mitogen-activated protein kinase JNK c-Jun N-terminal kinase TRAF6 tumor necrosis factor receptor-associated factor 6 SLE systemic lupus erythematosus RA rheumatoid arthritis IBD inflammatory bowel disease COPD chronic obstructive pulmonary disease CAAMR chronic active antibody-mediated rejection ALI acute lung injury GO gene ontology Q quartile AGP1 α-1-acid glycoprotein CXCL7 CXC chemokine ligand 7 Th T helper The analysis of protein-protein interactions indicated that the four proteins interacted with each other. Accord ing to the above points, we speculated that ORM1, SPARC, vWF and PPBP were involved in the TNF-α signalling pathway. ORM1 induced the NF-κB signal ling pathway and induced the production of inflamma tory cytokines, such as TNF-α and IL-6. Discussion In the KEGG pathway analysis, vWF was found participates in the complement and coagula tion cascades, which is well known. vWF was also found to participate in the ECM-receptor interaction, focal adhesion and PI3K-Akt signalling pathways, ultimately activating the NF-κB signalling pathway and MAPK sig nalling pathway; all of these pathways are associated with the TNF-α signalling pathway. Notably, Canobbio found that p38MAPK is phosphorylated upon platelet stimula tion by vWF [34]. Bernado showed that proinflammatory TNF-α can stimulate the release of vWF from endothe lial cells into the blood circulation [35].When TNF-α is increased, more vWF is released from endothelial cells. New findings have verified that vWF is associated with the inflammatory response in a direct or an indirect manner [33]. Nossent showed that the upregulation of vWF in SLE plays a role in immune-mediated inflamma tion, which is consistent with our hypothesis [36]. Simi lar to the case for ORM1, when vWF levels increased, the TNF-α signalling pathway was activated, thus inducing an immunological effect. f In the biological process cluster analysis, the regula tion of cell morphogenesis and secretion by cell terms were associated with SPARC, a secreted protein that is acidic and rich in cysteines. SPARC is a small molecu lar glycoprotein secreted by endothelial cells and fibro blasts that inhibits cell adhesion, regulates the cell cycle and promotes tissue fibrosis. It is closely related to cell proliferation and angiogenesis [37]. SPARC has been found to modulate transforming growth factor (TGF)-β Yin et al. Clinical Proteomics (2023) 20:24 (2023) 20:24 Page 9 of 11 Page 9 of 11 (2023) 20:24 Yin et al. Clinical Proteomics as a biomarker in lung cancer and advanced myelodys plastic syndrome [48, 49], suggesting that CXCL7 may participate in the progression of tumors. In the KEGG pathway analysis, PPBP was found to be participated in cytokine-cytokine receptor interactions and chemokine signalling pathways, which can indirectly modulate the MAPK signalling pathway. Wang showed that CXCL7 could be released by dendritic cells stimulated with TGF- β, although the function of TGF-β-induced CXCL7 was unknown. However, the findings support the view that CXCL7 may participate in the regulation of the immune response by inducing chronic inflammation or Th2 polarization [50]. Discussion vWF release was promoted by TNF-α, and vWF then participated in the PI3K-Akt signalling pathway, thus inducing the NF-κB and MAPK signalling pathways associated with the TNF-α signalling pathway. TNF-α reduced the produc tion of TGF-β via Smad6/7, and TGF-β bound to SPARC to reduce TNF-α levels. Downregulation of SPARC weak ened the reduction in TNF-α level in our study. CXCL7 (PPBP) could regulated TNF-α via IL-6, an important cytokine that is released downstream of the TNF-α sig nalling pathway, and IL-6 reduced the levels of CXCL7 (PPBP), while TGF-β induced CXCL7 expression. Similar to the case for SPARC, downregulation of PPBP weak ened the polarization of Th2 and may have induced the polarization of Th1 cells. In summary, the four proteins are all immune-related and are all involved in the TNF-α signalling pathway in the pathogenesis of ITP. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1186/s12014-023-09413-0. Supplementary Table S1: The KEGG-based functional enrichment analysis Supplementary Fig. 1 A: The KEGG pathway of PI3K-Akt Supplementary Fig. 1B: The TNF-α signalling pathway Supplementary Material 4: The Full MaxQuant results g Supplementary Table S1: The KEGG-based functional enrichment analysis Supplementary Fig. 1 A: The KEGG pathway of PI3K-Akt Supplementary Fig. 1B: The TNF-α signalling pathway Supplementary Material 4: The Full MaxQuant results Data Availability y All data generated or analysed during this study are included in this published article. 18. Radner H, Aletaha D. Anti-TNF in rheumatoid arthritis: an overview. Wien Med Wochenschr. 2015;165:3–9. 19. De la Cruz-Mosso U, García-Iglesias T, Bucala R, et al. MIF promotes a differ ential Th1/Th2/Th17 inflammatory response in human primary cell cultures: predominance of Th17 cytokine profile in PBMC from healthy subjects and increase of IL-6 and TNF-α in PBMC from active SLE patients. Cell Immunol. 2018;324:42–9. Funding 14. Xu X, Liu T, Yang J, et al. The First Whole-Cell Proteome- and lysine-acetylome- based comparison between Trichophyton rubrum Conidial and Mycelial Stages. J Proteome Res. 2018;17:1436–51. g The work was supported by grants from the Taishan Youth Scholar Foundation of Shandong Province (tsqn201812140), the Academic Promotion Program of Shandong First Medical University (2019RC018), Natural Science Foundation of Shandong Province (ZR2021MH319, ZR2020MH112), the National Natural Science Foundation of China (81570104), the Key Research and Development Project of Jinan (201907021; 201907026;202019160), Projects of medical and health technology development program of Shandong province(2019WS498). 15. Dimmer EC, Huntley RP, Alam-Faruque Y, et al. The UniProt-GO annotation database in 2011. Nucleic Acids Res. 2012;40:D565–D70. 16. Horton P, Park KJ, Obayashi T, et al. WoLF PSORT: protein localization predic tor. Nucleic Acids Res. 2007;35:W585–W7. 17. Szklarczyk D, Franceschini A, Kuhn M, et al. The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored. Nucleic Acids Res. 2011;39:D561–D8. Received: 9 November 2021 / Accepted: 3 June 2023 Received: 9 November 2021 / Accepted: 3 June 2023 Received: 9 November 2021 / Accepted: 3 June 2023 24. Parameswaran N, Patial S. Tumor necrosis factor-α signaling in macrophages. Crit Rev Eukaryot Gene Expr. 2010;20:87–103. https://doi.org/10.1615/critrev eukargeneexpr.v20.i2.10. PMID: 21133840; PMCID: PMC3066460. 25. Schmid K, Nimberg RB, Kimura A, Yamaguchi H. THE CARBOHYDRATE UNITS OF HUMAN PLASMA al-ACID GLYCO- PROTEIN. Biochim Biophys Acta. 1977;492:291–302. 26. Fournier T, Medjoubi-N N, Porquet D. Alpha-1-acid glycoprotein. Biochim Biophys Acta. 2000;1482:157–71. Consent for publication Not applicable. 22. Zhang F, Chu X, Wang L, et al. Cell-mediated lysis of autologous plate lets in chronic idiopathic thrombocytopenic purpura. Eur J Haematol. 2006;76:427–31. Conclusion Authors contribution YDM and YD contributed to experimental design and data analysis; SRJ and SXH contributed to data analysis and writing of the article; XHZ and HSY contributed to the statistical analysis; SNN obtained funding, experimental design and writing of the article. All authors read and approved the final article. In general, this study provides a multilevel proteomic data resource for ITP research and increases under standing of the immunological function of ITP. The pro teomic landscape of ITP is highly informative, identifying specific proteins. In fact, our comprehensive analysis Page 10 of 11 Yin et al. Clinical Proteomics (2023) 20:24 Yin et al. Clinical Proteomics (2023) 20:24 Yin et al. Clinical Proteomics (2023) 20:24 References 1. Cines DB, Bussel JB, Liebman HA, Luning Prak ET. The ITP syndrome: patho genic and clinical diversity. Blood. 2009;113:6511–21. 1. Cines DB, Bussel JB, Liebman HA, Luning Prak ET. 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Increased von willebrand factor levels in patients with systemic lupus erythematosus reflect inflammation rather than increased propensity for platelet activation. Lupus Sci Med. 2016;3:e000162. 10. Mallick P, Kuster B. Proteomics: a pragmatic perspective. Nat Biotechnol. 2010;28:695–709. 11. Rauniyar N. Parallel reaction monitoring: a targeted experiment performed using high resolution and high Mass Accuracy Mass Spectrometry. Int J Mol Sci. 2015;16:28566–81. 37. Brekken RA, Sage EH. SPARC, a matricellular protein: at the crossroads of cell– matrix communication. Matrix Biol. 2001;19:815–27. 12. Lambert MP, Gernsheimer TB. Clinical updates in adult immune thrombocy topenia (ITP). Blood. 2017;129:2829–35. 38. Francki A, McClure TD, Brekken RA, et al. SPARC regulates TGF-beta1- dependent signaling in primary glomerular mesangial cells. J Cell Biochem. 2004;91:915–25. 13. Liu SY, Yuan D, Sun RJ, Zhu JJ, Shan NN. Significant reductions in apoptosis- related proteins (HSPA6, HSPA8, ITGB3, YWHAH, and PRDX6) are involved in immune thrombocytopenia. J Thromb Thrombolysis. 2021;51:905–14. https:// doi.org/10.1007/s11239-020-02310-5. Epub 2020 Oct 12. PMID: 33047245. 13. Liu SY, Yuan D, Sun RJ, Zhu JJ, Shan NN. Significant reductions in apoptosis- related proteins (HSPA6, HSPA8, ITGB3, YWHAH, and PRDX6) are involved in immune thrombocytopenia. J Thromb Thrombolysis. 2021;51:905–14. https:// doi.org/10.1007/s11239-020-02310-5. Epub 2020 Oct 12. PMID: 33047245. Page 11 of 11 Yin et al. Publisher’s Note 46. Yeo L, Adlard N, Biehl M, et al. Expression of chemokines CXCL4 and CXCL7 by synovial macrophages defines an early stage of rheumatoid arthritis. Ann Rheum Dis. 2016;75:763–71. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
W4292585131.txt	https://zenodo.org/record/6369741/files/766-767.pdf	en		Oxidation of Aliphatic Ketones by Ditellurato Argentate(III) in Alkaline Medium-A Kinetic Study	Zenodo (CERN European Organization for Nuclear Research)	1,981	cc-by	982	J. Indian Cbem. Soc., Vol. LVDI, August 1981, pp. 766-767 Oxidation of Aliphatic Ketones by Ditellurato Argentate(III) In Alkaline Medium-A Kinetic Study T. RAVI PRASAD, B. SETHURAM and T. NAVANEETH RAO Department of Chemistry, Osmanla University, Hyderabad-500 007 Manuscript received 26 June 1980, revised 2 February 1981, accepted 19 Mayl981 Kinetics of oxidation of acetone, methyl ethyl, methyl n-propyl, methyl iso-propyl and methyl t-butyl ketones by ditellurato argentate(lll) in alkaline medium was investigated. Tbe kinetic data and product analysis indicate that only the keto form of the substrate takes part in these reactions. A suitable mechanism consistent with tbe observed rate law is postulated. Taft equation was found to b(! applicable for the series witb p • = 3.00. T HE mechanism of oxidation of ketones using various oxidants has been the subject of study by several workers. Two different views on the form of ketone (keto or enol) involved in the reaction have been expressed. Since the relative •rates of enolization and oxidation were the same in oxidations by Cr(VI) 1 and ferricyanide 11 it was suggested that these oxidants attack the enol form. In the case of peroxy-diphosphate-ketone reaction in acid mediums and periodate-ketone reaction in alkaline medium4,, the rate of oxidation was found to be very much greater than that of enolization. It was therefore concluded that in these reactions the attack is on the keto form. In view of this it was thought worthwhile to investigate which one of the tautomers is involved in the reaction during the oxidation of aliphatic ketones by ditellurato argentate(III). The chemicals used were of AR grade and purified wherever necessary. Ditellurato argentate(III) (DT A) solution was prepared and estimated as reported earlier 11 • The progress of reaction was followed spectrophtometrically by finding the absorbance of Ag(III) solutions at 395 nm. Stoichiometric studies revealed the reactions to be of 1 : 1 type. Under the experimental conditions the main product identified was acetic acid. Under the conditions (Ag(III)]<<[ketone], a plot of log (absorbance) vs time was linear mdicating the order in [Ag(III)] to be unity. From the slopes of such plots the pseudo-first order rate constants (k') were calculated. The plot of log k' vs log (ketone] was also found to be linear in each case indicating the order in {ketone] to be also unity. The rate of oxidation was found to increase with an increase in [OH-] and decreased with an increase in [tellurate]. For instance, in the reaction with acetone at constant [Ag(III)], (acetone] and temperature (304K) the rate constant increased from 1.34 X 10-s min- 1 to 3.28 x w-s min- 1 when (OH-] was increased from 3.00 x IQ-s M to 6.00 x I0- 11 M. Under the same conditions at constant (OH-] the rate constant 766 decreased from 2.97 X 1o-Il m in- 1 to 0.900 X IO-S min- 1 when [tellurate] was increased from 7.5 x w-&M to 25.0x w-•M. No free radicals were detected in these reactions. The bimolecular rate constants for these oxidation reactions (Table I) are very high (10 8 to 10• times) when compared with the corresponding enolization rates of the ketones 6 ·'~' under the same experimental conditions. We therefore assume.the keto form to be the reactive species in the case of all the ketones studied. The probable mechanism of oxidation appears to be : - K1 [Ag(OH) 11 (H 4 Te0 6 )(H~ Te0 6 ))R-+OH~ (Ag(OH) 11 (H 8 Te0 8 )](H~ Te0 6 )]•-+H 11 0 ... (I) Ks (Ag(OH) 11 (H 8 TeO.,)](H4, Te0 6 )]•-~ [Ag(OH) 1 (H 4 TeO.,)]-+(H 8 Te0 6 ) 8 ... (2) r k· 8 '-., H C C=O+Ag(III)~~ / slow R a-'-. ~ H 0 c.. C=O~Ag / R 8 -+ fast CH9COOH+Ag(I)+Other products ... (3) Equilibria (1) and (2) are written in the light of the arguments reported in our earlier paper•. The rate law for the above scheme comes out to be - d[Ag(III)] = K 1 K 11 k 8 [Ag(III)]rRCOCH 8 ][0H-l dt [(H 8 Te0 6 ) 8 - ] which explains well all the kinetic data obtained. It can be seen from Table l that the order of reactivity is acetone>methyl ethyl ketone>methyl n-propyl ketone>methyl iso-propyl ketone>metbyl t-butyl ketone, which is also the order of ketone content. This points out a probable attack on the ketonic form by Ag(III) and supports the view that the oxidation does not involve electron transfer from the enolic form though the medium is basic. The sa me trend of reactivity was reported by Panigrahi et a/ 8 in the oxidation of aliphatic ketones PRASAD, SBTHURAM &, RAO OXIDATION OF ALIPHATIC KETONES BY DITELLURATO ETC. TABI.lt 1-BIMOI.ECUI.AR RATE CONSTANTS FOR Aga+-K:RTON!t REACTION Acetone k" at 804"K dm•moi- 1 min- 1 1.76 methyl ethyl ketone 0.888 methyl n-propyl ketone methyl iso-propyl ketone 0.460 by periodate in alkaline medium. Taft's plot of log k/ko vs a* was found to be linear (correlation coefficient= 0.997) with a slope, p* = 3.00. The positive value of P'* suggests an electron-rich ttransition state and therefore the reaction rate must decrease with the electron releasing capacity of the groups as can be seen from the k" values in Table I. References 1. B. K. TANDON, K. K. BENARJI and G. V. BAKORE, Indian J. Ohem., 1971, 9, 677. 0.360 methyl ter-butyl ketone 0.215 2. V. N. SINGH and M.P. SINGH, Indian J, Ohem., 1970, 8, 3. 1\L SANTAI'l'A and P. MARUTBAMUTHU, Indian J, Ohem., 529. 1974, 12, 424. T. RAVI PRASAD, B. 8:RTBURAM and T. NAVAN:RKTH RAO, Indian J. Ohem., 1979, 18A, 40. 5. P. K. JAISWAI. and K. L. YADAV, Talanta, 1970,7, 236. 6. A. MEKNAKSHI and M. BANTA PI' A, J. Oatalflsis, 1970, 10, 4. 7. 8. 300. • R. P. B:ax.x., J, Ohem. Soc (B). 1966, 241 B. G. P. PANIGRABI and P. K. MISRA, Indian J, Ohem., 1978, 16A, 762. 161
https://openalex.org/W3159315854	https://eejournal.ktu.lt/index.php/elt/article/download/28841/14818	English	null	Textile Antenna for First-Person View Goggles	Elektronika ir elektrotechnika	2,021	cc-by	4,121	Manuscript received 1 February, 2021; accepted 6 April, 2021. This work is funded by FCT/MCTES through national funds and when applicable co-funded EU funds under Grant No. UIDB/50008/2020- UIDP/50008/2020 and Grant No. UIDB/00195/2020. This work is also funded by the Fundação para a Ciência e Tecnologia (FCT) through Fundo Social Europeu (FSE) and by Programa Operacional Regional do Centro under the PhD Grant No. SFRH/BD/139847/2018. Textile Antenna for First-Person View Goggles Luis Andre1, 2, , Pedro Pinho1, 2, Carolina Gouveia1, 3, Caroline Loss4 1Instituto de Telecomunicacoes, Aveiro, Portugal 2Instituto Superior de Engenharia de Lisboa, Lisboa, Portugal 3Universidade de Aveiro, Aveiro, Portugal 4FibEnTech Research Unit, Covilha, Portugal a44681@alunos.isel.pt Luis Andre1, 2, , Pedro Pinho1, 2, Carolina Gouveia1, 3, Caroline Loss4 1Instituto de Telecomunicacoes, Aveiro, Portugal 2Instituto Superior de Engenharia de Lisboa, Lisboa, Portugal 3Universidade de Aveiro, Aveiro, Portugal 4FibEnTech Research Unit, Covilha, Portugal a44681@alunos.isel.pt To have a functional FPV system, three components are needed: 1Abstract—Unmanned Aerial Vehicles, also known as drones, are vehicles controlled remotely. First-Person View (FPV) technology allows these vehicles to have greater versatility and be more easily piloted. The antennas currently used for video transmission are bulky and uncomfortable, so there is a need for this volume to be reduced to increase portability, being this the focus of the paper. The antenna was developed in textile materials to reduce its size and weight, as these materials allow for a better comfort and a reduced footprint. This paper presents a microstrip array adapted for the frequencies used in FPV video transmission. The designed textile antenna was compared with a commercially available rigid antenna used for this transmission. The comparison shows promising results for the antenna made of textile materials. The main goal of increasing the comfort and portability of the FPV goggles was achieved. 1. A drone, which is the aircraft that will be piloted; 2. A controller that allows the pilot to control the drone; 3. The FPV goggles that allow the pilot to view what is captured by the drone’s camera, as shown in Fig. 1. 3. The FPV goggles that allow the pilot to view what is Observing Fig. 1, it is also possible to see that the FPV goggles have two different receiver antennas, an omnidirectional and a directional one, to ensure that the video reception will be continuous independent of the drone’s position (transmitter antenna). Fig. 1. Complete FPV System. Index Terms—Antennas; Array antennas; Drones; FPV; Textile Antenna. ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 http://dx.doi.org/10.5755/j02.eie.28841 I. INTRODUCTION Unmanned Aerial Vehicles (UAVs), also known as drones, are aerial vehicles that can be controlled remotely and do not require a crew on board. With the increasing demand and interest in drones, the First-Person View (FPV) technology appears. FPV is a method used to pilot drones that allows the pilot to get a perspective as if he was inside the aircraft, thus making it possible to obtain a better control and a more useful perspective for those who fly the aircraft. This technology opens possibilities in several areas, such as intervention and rescue in dangerous environments [1]. In addition, in agriculture, e.g., allowing the farmer to observe agricultural fields more efficiently [2]. This technology can also be used in addition to virtual reality, for tourism marketing, exploring different places around the world in real-time [3]. Despite the number of applications, nowadays, the most explored fields on FPV are surveillance and Drone Racing competitions [4]. Fig. 1. Complete FPV System. In practice, this technology requires a connection between a display, which will be the FPV goggles, and the camera onboard of the drone that will transmit the image via a video transmitter. Currently, this connection is made by antennas typically built on a rigid substrate, such as FR-4. The low- cost antennas for FPV can achieve larger sizes, as shown in Fig. 2, and hence significantly increase the weight of the FPV goggles, thus decreasing their wearability. The antenna shown in Fig. 2 is the low-cost Triple Feed Patch Antenna by Maarten Baert [5], and it is widely used in the FPV community. Both the problems mentioned before - the larger size and the added weight, combined with how these antennas connect to the goggles, make them often hang, 49 ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 resulting in them being loosely secured. This issue makes them hard to wear, especially if the user is continuously moving or changing his head position. antennas requires the characterization of their properties. Specific electrically conductive textiles are available on the market and have been successfully used, and ordinary textile fabrics have been used as substrates. Fig. 2. Example of a rigid antenna currently used in an FPV system [2]. Beyond choosing the textile materials, the construction technique of the antenna is also crucial because the textile materials are highly deformable. II. TEXTILE ANTENNAS The wearable antenna is the key to make electronic devices less obtrusive. This will enhance the interaction of the user with some electronic devices, making them less invasive and more discrete. Since 2001, when P. Salonen, M. Keskilammi, J. Rantanen, and L. Sydanheimo [7] proposed the first prototype of flexible antenna made of fabrics, the use of textile materials to develop wearable antennas has increased exponentially. I. INTRODUCTION The geometrical dimensions of the conductive patch and of the dielectric substrate should remain stable when connecting them, as the mechanical stabilization of both materials is essential to preserve the desired characteristics of the antenna [8]. Several techniques have been explored to manufacture textile antennas, such as laminating [11]–[13], sewing [14], embroidering [15]–[16], and screen printing [17]–[18]. Although textile antennas are used in several applications, no studies were found regarding the FPV ones. Even in the context of rigid antennas for FPV, the only studies found are regarding the ground station’s antennas, for instance [19]. In this way, this paper proposes a new application for wearable antennas by designing and testing a textile array for FPV goggles. Fig. 2. Example of a rigid antenna currently used in an FPV system [2]. In this paper, an innovative approach is proposed to increase the FPV User Experience (UX). The UX is described as the overall experience of a person using a product, especially in terms of how easy or pleasing it is to use [6]. For this, the use of textile antennas is proposed to reduce the weight of the FPV goggles without reducing the antenna’s performance. In this way, it will be possible to increase the comfort of the FPV goggles and to solve the wearability issues. A. FPV Standards FPV system antennas must follow a few standards, according to [4]. The frequencies used for FPV transmission are within a range from 5.645 GHz to 5.945 GHz, where 5.795 GHz is the central frequency. Therefore, the designed antenna should cover all bandwidth [5]. This paper will focus on designing the directional antenna presented in the FPV system (see Fig. 1). Therefore, to achieve the desired directivity, a 2×2 antenna array will be used. The realized gain of this type of array is expected to be between 9 dBi and 12 dBi. The antenna will also have circular polarization, as it is the most common polarization in FPV systems. The transmitting antenna is located on the drone, which is constantly moving in relation to the reception antenna located on the FPV goggles. In this sense, circularly polarized antennas can increase the received signal energy under these conditions. The polarization chosen for the designed antenna was Right Hand Circular Polarization (RHCP). Firstly, the antenna was designed having in consideration the materials used for its construction. Then, the performance of the designed textile antenna was compared with a commercially available rigid antenna. This paper is organized as follows. First, a brief overview about textile antennas is given in Section II. The antenna design is presented in Section III. The results of the built antenna are presented in Section IV. Then follows Section V comparing the results of the built antenna with an antenna commercially available for FPV. Conclusions are presented in Section VI. IV. ANTENNA RESULTS To test the performance of the designed array, the textile antenna was manufactured and measured. The textile antenna array was manufactured through the laminating manufacturing technique, where the conductive and dielectric layers of textiles are bonded together using a thermally activated adhesive sheet. In this case, a 100 % polyamide continuous web was used. Furthermore, a Kapton tape was used to stabilize the radiating part of the array, ensuring that none of the lines would deform or move out of place during the laminating process. To ensure the geometric dimensions of the antenna, both textiles (conductive and dielectric ones) were cut using a laser cut machine. Figure 5 shows the manufactured array. Fig. 3. Single patch design. Fig. 3. Single patch design. ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 used. Therefore, the array substrate and ground plane had a size of 80 mm × 80 mm to fit in front of the goggles. The final array dimensions are shown in Table I, where l is the length and w is the width of the line. material’s dielectric properties were characterized at 5.8 GHz, using a resonant-based technique [10]. For the conductive parts, patch and ground plane, the Pure Copper Polyester Taffeta Fabric was used. This conductive material is a 100 % polyester fabric coated with copper and has a thickness of 0.08 mm and a conductivity of 62500 S/m. Fig. 4. Full array design. C. Single Element Radiation To design the full textile array, the first step was to simulate and build a single radiating element. The simulation was made using CST Studio Suite Software. To obtain circular polarization, the upper right and lower left corners were cut out. To achieve the desired resonance frequency and the best axial ratio possible, two parameters were varied, namely, the length of the patch L, being this the same as its width and the chamfer length e. To check the input impedance and the axial ratio, a line with half wavelength of length was used, as shown in Fig. 3. Fig. 3. Single patch design. Fig. 4. Full array design. TABLE I. DIMENSIONS OF THE DESIGNED ANTENNA (UNITS: mm) TABLE I. DIMENSIONS OF THE DESIGNED ANTENNA (UNITS: mm). L e L1 L2 l w l w 16.89 5.44 4.71 1.31 35.60 1.31 L3 L4 L5 l w l w l w 11.00 3.02 5.64 0.88 3.10 3.00 B. Design Considerations As mentioned in Section I, currently, the commercially available antennas for FPV video reception are made of rigid materials. With the advancement in FPV technology, one of the biggest trends is the increasing portability of the whole system. However, this fact is not observed in the antennas used for video reception. To increase the UX, textile materials were chosen for the antenna construction. Compared with the commercially available antennas, the textile material allows the designed antenna to have a smaller size and a more comfortable utilization since the textile is lighter and more flexible material than the rigid ones. To achieve good results, wearable antennas have to be thin, lightweight, low maintenance, robust, low-cost, and easily integrated in radio frequency circuits [8]. In this way, planar antennas have been proposed for textile applications because they fulfil all these requirements and are adaptable to any surface [9]. To develop this type of antenna, the knowledge of the properties of textile materials is crucial, as well as the knowledge of the manufacturing techniques. Several properties of the materials influence the behavior of the antenna [10]–[11]. Thus, the use of textiles in wearable To design the textile antenna array, commercially available materials were selected and characterized. For the dielectric substrate, the PDE Black (Borgstena Textile Portugal) was chosen. It is a weft-knit fabric with 0.75 mm of thickness, ϵr = 1.385, and the loss tangent is of 0.008. The 50 D. Full Array Simulation To design the array feeding network, the input impedance of the single radiating elements has to be taken into consideration to assure proper impedance matching. The dimensioning of the feeding network was specially using the smallest number possible of quarter wavelength transformers since they lead to a reduced bandwidth. Furthermore, the usage of thinner lines helps to reduce the radiation effects of the lines. However, it is also important to keep in mind the construction limits. The upper part of the array feeding network consists of a line that connects both radiating patches horizontally. This line has an impedance approximate to the input impedance of the patch, which is approximately 70 Ω, and it is defined as L1 and L2 lines in Fig. 4. The length of this line defines the spacing between the radiating elements, which in this case is ¾ λ. This spacing allows a balanced trade-off between antenna parameters, such as gain, half power bandwidth (HPBW), and side-lobe level. The upper part is also connected to a quarter-wavelength transformer identified as L3, with the necessary width to allow the impedance to be transformed from 35 Ω to the desired 100 Ω, which is the impedance of the line L4. This line connects the lower part of the array, which is similar to the upper part. Finally, both the upper and lower parts connect to a 50 Ω impedance line L5 where an SMA connector was added. The final size of the array had to be adjusted due to the size limitations of the goggles Fig. 5. Manufactured textile array. Fig. 5. Manufactured textile array. After the construction, the performance of the antenna was analysed. The S11 parameter was measured in a 51 ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 laboratory environment, using a Vector Network Analyzer (VNA) from Anritsu model MS4647B shown in Fig. 6. laboratory environment, using a Vector Network Analyzer (VNA) from Anritsu model MS4647B shown in Fig. 6. Telecomunicações e Informática from Aveiro University. Figs. 7–9 present the measured results. In Fig. 7, it is possible to observe the measured and simulated results of the S11 parameter. The simulated results show that the S11 is lower than -10 dB from 5.473 GHz to 6.260 GHz, and it is equal to -38.91 dB at the central frequency. D. Full Array Simulation The manufactured antenna presented a significant increase in the bandwidth, which it is now between 5.392 GHz and 6.545 GHz, resulting in a bandwidth of 1.153 GHz. The S11 at the frequency of interest (5.795 GHz) was equal to -17.48 dB. Fig. 6. VNA Anritsu MS4647B used for measurements. The radiation pattern was measured in anechoic chamber provided by DETI - Departamento de Eletronica, The antenna gain was also measured being equal to 6 dBi, while the simulated one was equal to 9.04 dBi. This lower value can be due to the antenna manufacturing process, as it is handmade or also due to high losses inherent to the textile materials. Figure 8 shows the normalized radiation diagram at ϕ = 0 plane of both the manufactured and the simulated antenna. It is possible to extract the half-power beam width (HPBW) that for the simulated antenna was 36 º and the manufactured antenna was approximately 40 º. Regarding the axial ratio, the simulated one was equal to 0.32 dB at the central frequency. Fig. 6. VNA Anritsu MS4647B used for measurements. Fig. 6. VNA Anritsu MS4647B used for measurements. The radiation pattern was measured in anechoic chamber provided by DETI - Departamento de Eletronica, Fig. 7. Comparison of the simulated and measured S11 parameter of the textile antenna array. Fig. 8. Comparison of the simulated and measured normalized radiation pattern of the textile antenna array. Fig. 7. Comparison of the simulated and measured S11 parameter of the textile antenna array. Fig. 7. Comparison of the simulated and measured S11 parameter of the textile antenna array. Fig. 8. Comparison of the simulated and measured normalized radiation pattern of the textile antenna array. Fig. 8. Comparison of the simulated and measured normalized radiation pattern of the textile antenna array. adapted for the desired frequencies. The commercial antenna has a S11 lower than -10 dB from 5.214 GHz to 6.302 GHz, resulting in a bandwidth of 1.088 GHz, and at the centre frequency, has a value equal to -15.76 dB. When comparing both antennas, it is possible to observe that the designed textile antenna has a better bandwidth and lower S11 value at the central frequency. prototype allowing the antenna to be covered and discreet. prototype allowing the antenna to be covered and discreet. (a) (b) Fig. 10. Physical comparison between (a) commercial and (b) manufactured antennas. (b) ( ) REFERENCES [1] A. Saha, A. Kumar, and A. K. Sahu, “FPV drone with GPS used for surveillance in remote areas”, in Proc. of 3rd International Conference on Research in Computational Intelligence and Communication Networks (ICRCICN), Kolkata, India, 2017, pp. 62–67. DOI: 10.1109/ICRCICN.2017.8234482. [2] V. Puri, A. Nayyar, and L. Raja, “Agriculture drones: A modern breakthrough in precision agriculture”, Journal of Statistics and Management Systems, vol. 20, no. 4, pp. 507–518, 2017. DOI: 10.1080/09720510.2017.1395171. (a) (b) Fig. 10. Physical comparison between (a) commercial and (b) manufactured antennas. (a) [3] U. Stankov, J. Kennell, A. M. Morrison, and M. D. Vujičić, “The view from above: The relevance of shared aerial drone videos for destination marketing”, Journal of Travel & Tourism Marketing, vol. 36, no. 7, pp. 808–822, 2019. DOI: 10.1080/10548408.2019.1575787. V. COMPARISON WITH A COMMERCIAL ANTENNA This section aims to compare the designed textile antenna with an antenna that is widely used by the FPV Drone community. The antenna used in this comparison is the Triple Feed Patch Antenna by Maarten Baert (see Fig. 2), which is made with the FR-4 substrate. Figure 9 presents the comparison of the measured S11 parameters between the designed antenna and the commercially available one. Table II shows the key parameters of both antennas. As shown, the proposed textile antenna has competitive results in almost all aspects. The only result that differs between In Fig. 9, it is possible to observe that both antennas are 52 CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest. The authors declare that they have no conflicts of interest. ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 them is the gain, where the designed antenna is slightly lower than the commercial. This is an expected result since the textiles are very lossy materials due to their high porosity. Furthermore, both antennas were compared in terms of physical properties. Comparing both antennas side by side, as seen in Fig. 10, the designed antenna has a much smaller footprint than the commercial one. In fact, the designed antenna has 80 mm × 80 mm against the 120 mm of diameter of the commercial one. In addition, the designed textile antenna is 17 g lighter. Furthermore, the way it is placed in the goggles improves the user’s mobility and comfort, which is unattainable with the currently available antennas. The combination of all these factors creates a highly competitive antenna capable of competing with the antennas currently available for FPV systems. TABLE II. TABLE WITH THE COMPARISON RESULTS. Commercial Antenna Designed Antenna Central Frequency 5.8 GHz 5.795 GHz Bandwidth 1.088 GHz 1.153 GHz S11 () -15.76 dB -17.46 dB Antenna Gain 9.3 dBi () 6.0 dBi HPBW 55 º () 40 º Note: () At the central frequency; (*) These values were taken from the official page of the antenna, as these were not measured [3]. TABLE II. TABLE WITH THE COMPARISON RESULTS. Commercial Antenna Designed Antenna Central Frequency 5.8 GHz 5.795 GHz Bandwidth 1.088 GHz 1.153 GHz S11 () -15.76 dB -17.46 dB Antenna Gain 9.3 dBi () 6.0 dBi HPBW 55 º () 40 º Note: () At the central frequency; () These values were taken from the official page of the antenna, as these were not measured [3]. Note: () At the central frequency; (**) These values were taken from the official page of the antenna, as these were not measured [3]. Fig. 9. Comparison of the S11 parameter between the manufactured textile antenna and the commercial one. Fig. 9. Comparison of the S11 parameter between the manufactured textile antenna and the commercial one. prototype allowing the antenna to be covered and discreet. VI. CONCLUSIONS [4] J. Delmerico, T. Cieslewski, H. Rebecq, M. Faessler, and D. Scaramuzza, “Are we ready for autonomous drone racing? The UZH- FPV drone racing dataset”, in Proc. of International Conference on Robotics and Automation (ICRA), Montreal, 2019, pp. 6713–6719. DOI: 10.1109/ICRA.2019.8793887. In this work, a solution to the current problems with portability and comfort of FPV antennas was presented. The solution was to use textile antennas instead of the typical rigid substrate. The designed antenna was compared with a commercially available one. This comparison shows promising results for the designed antenna and achieving portability and comfort that cannot be achieved by the antennas currently available while maintaining a gain of 6 dBi and a bandwidth of 1.153 GHz centred at 5.795 GHz. The antenna directivity was also maintained achieving a value of HPBW of 40 º. The footprint of the designed antenna was also reduced having the designed antenna size of 80 mm × 80 mm. In future work, we intend to explore different types of textile materials, more mechanically stable, allowing for better preservation of the antenna’s characteristics and the development of a goggles cover [5] “Triple Feed Patch antenna - Quadcopters - Maarten Baert’s website”, Maartenbaert.be, 2021. [Online]. Available: https://www.maartenbaert.be/quadcopters/antennas/triple-feed-patch- antenna/ [6] R. Polacek, “User interface concept for smart glasses”, BSc. thesis, University of Economics in Prague, May 2020. University of Economics in Prague, May 2020. [7] P. Salonen, M. Keskilammi, J. Rantanen, and L. Sydanheimo, “A novel bluetooth antenna on flexible substrate for smart clothing”, in Proc. of IEEE Int. Conf. Syst. Man, Cybern., Tucson, AZ, USA, 2001, pp. 789–794, vol. 2. DOI: 10.1109/ICSMC.2001.973011. [8] R. Salvado, C. Loss, R. Goncalves, and P. Pinho, “Textile materials for the design of wearable antennas: A survey”, Sensors, vol. 12, no. 11, pp. 15841–15857, 2012. DOI: 10.3390/s121115841. [9] C. A. Balanis, Antenna Theory: Analysis and Design, 3rd ed., Wiley Interscience: Hoboken, NJ, USA, 2005. [10] C. Loss, R. Gonçalves, P. Pinho, and R. Salvado, “Influence of some structural parameters on the dielectric behaviour of materials for 53 ELEKTRONIKA IR ELEKTROTECHNIKA, ISSN 1392-1215, VOL. 27, NO. 2, 2021 textile antennas”, Textile Research Journal, vol. 89, no. 7, pp. 1131– 1143, 2018. DOI: 10.1177/0040517518764000. [15] A. Tsolis, W. G. Whittow, A. A. Alexandridis, and J. C. Vardaxoglou, “Embroidery and related manufacturing techniques for wearable antennas: Challenges and opportunities”, Electronics, vol. 3, no. 2, pp. 314–338, 2014. DOI: 10.3390/electronics3020314. [11] C. Loss, R. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution 4.0 (CC BY 4.0) license (http://creativecommons.org/licenses/by/4.0/). VI. CONCLUSIONS Salvado, R. Gonçalves, and P. Pinho, “Influence of the laminating manufacturing technique on the Sn parameter of printed textile antennas”, in Proc. of 2017 IEEE MTT-S International Microwave Workshop Series on Advanced Materials and Processes for RF and THz Applications (IMWS-AMP), Pavia, Italy, 2017, pp. 1– 3. DOI: 10.1109/IMWS-AMP.2017.8247426. 314–338, 2014. DOI: 10.3390/electronics3020314. [16] A. Kiourti, C. Lee, and J. L. Volakis, “Fabrication of textile antennas and circuits with 0.1 mm precision”, IEEE Antennas and Wireless Propagation Letters, vol. 15, pp. 151–153, 2016. DOI: 10.1109/LAWP.2015.2435257. [12] C. Hertleer, H. Rogier, L. Vallozzi, and L. Van Langenhove, “A textile antenna for off-body communication integrated into protective clothing for firefighters”, IEEE Trans. Antennas Propag., vol. 57, no. 4, pp. 919–925, 2009. DOI: 10.1109/TAP.2009.2014574. [17] M. L. Scarpello, I. Kazani, C. Hertleer, H. Rogier, and D. Vande Ginste, “Stability and efficiency of screen-printed wearable and washable antennas”, IEEE Antennas Wirel. Propag. Lett., vol. 11, pp. 838–841, 2012. DOI: 10.1109/LAWP.2012.2207941. [13] C. Loss, R. Gonçalves, C. Lopes, P. Pinho, and R. Salvado, “Smart coat with a fully-embedded textile antenna for IoT applications”, Sensors, vol. 16, no. 6, 2016. DOI: 10.3390/s16060938. [18] W. G. Whittow et al., “Inkjet-printed microstrip patch antennas realized on textile for wearable applications”, IEEE Antennas and Wireless Propagation Letters, vol. 13, pp. 71–74, 2014. DOI: 10.1109/LAWP.2013.2295942. [14] P. Salonen, Y. Rahmat-samii, M. Schaffrath, and M. Kivikoski, “Effect of textile materials on wearable antenna performance: A case Study of GPS antenna”, in Proc. of IEEE Antennas and Propagation Society Symposium (APS), 2004, pp. 459–462, vol. 1. DOI: 10.1109/APS.2004.1329673. [19] M. Martínez-Vázquez, “Low-cost RHCP array at 5.8 GHz for FPV ground stations”, in Proc. of 2013 IEEE Antennas and Propagation Society International Symposium (APSURSI), Orlando, FL, USA, 2013, pp. 2227–2228. DOI: 10.1109/APS.2013.6711772. 54

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